Yeast, yeast extract containing gamma-glu-abu, and a method for producing the same

ABSTRACT

A yeast extract containing 0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract is produced by culturing a yeast, such as  Saccharomyces cervisiae  or  Candida utilis , in a medium containing a compound selected from Abu (L-2-aminobutyric acid) and γ-Glu-Abu (L-γ-glutamyl-L-2-aminobutyric acid), and preparing a yeast extract from the obtained cells.

TECHNICAL FIELD

The present invention relates to a yeast and yeast extract containingγ-Glu-Abu (L-γ-glutamyl-L-2-aminobutyric acid), as well as a method forproducing the same. The yeast extract of the present invention is usefulin the field of foodstuffs such as seasonings and health foods.

BACKGROUND ART

Yeast extracts have a function of imparting atsumi (thickness), umami,etc. to foodstuffs, and have been widely used as seasonings in the fieldof foodstuffs. Especially, glutathione (henceforth also referred to as“GSH”), which is a tripeptide consisting of glutamic acid, cysteine andglycine, is known to impart kokumi to foodstuffs (Non-patent documents 1and 2), and seasonings containing GSH have been developed.

Meanwhile, although the calcium sensing receptor (CaSR), which is aG-protein classified into the class C, has been reported to respond toGSH (Non-patent document 3), the physiological significance thereof hasnot been clarified. Moreover, this CaSR is present also in the lingualcells, and it was considered to show a certain taste response(Non-patent document 4). Then, it has recently been clarified that thisCaSR participates in recognition of kokumi in humans (Non-patentdocument 5). This reference reported that not only GSH that has beenrecognized as a kokumi substance, but also several γ-glutamyl compoundssimilarly respond to CaSR. Furthermore, it has been reported thatpeptides represented by the general formula γ-Glu-X or γ-Glu-X-Gly (Xcan represent an amino acid or amino acid derivative other than Cys),for example, γ-Glu-Met, γ-Glu-Thr, γ-Glu-Val-Gly, etc. have akokumi-imparting effect (Patent document 1). Moreover, the group ofesters including S- or O-carboxyalkylated γ-glutamyl or β-asparagylpeptides, and so forth are also reported as kokumi substances (Patentdocument 2). Although these peptides impart kokumi to foodstuffs likeGSH, they do not have a reduced SH group unlike GSH. It is known that asubstance having the reduced SH group such as GSH is generally unstable,and titer thereof is reduced with formation of disulfide bond (Patentdocument 2). However, γ-Glu-X, γ-Glu-X-Gly etc. are considered usefulfrom the viewpoint that the kokumi-imparting peptides not having thereduced SH group are stable.

Tastes sensed after eating change with time, and the tastes are calledinitial taste, middle taste, and aftertaste in the order from the tastesensed immediately after eating. Although tastes imparted by varioussubstances change with time in various patterns, concerning especiallykokumi, a kokumi-imparting substance showing a taste-imparting patternthat imparts strong initial taste is highly desired. It is known thatthe γ-glutamyl compound, γ-Glu-Abu-Gly, has a kokumi-imparting actionthat mainly imparts initial-middle taste (Patent documents 3 and 4).

It is known that the synthesis and decomposition of glutathione, whichis one of the γ-glutamyl compounds, is catalyzed by several enzymeswhich make up the γ-glutamyl cycle. In particular, γ-glutamyltranspeptidase is known to transfer the glutamate of GSH at theγ-position to another compound having an amino group, resulting indecomposition of GSH to cysteinylglycine (Non-patent document 6). It isconsidered that, if the compound having an amino group in this reactionis an amino acid, a dipeptide of γ-Glu-X can be generated as aby-product. However, research about producing these compoundseffectively using microorganisms has not been positively performed todate, partially because they are by-products.

An analysis of the fermentation broth of Micrococcus glutamicus can benoted as findings about the dipeptide γ-Glu-X (Non-patent document 7).This reference reported that the fermentation broth was loaded ontovarious columns to separate peptides and the like, resulting in theisolation of γ-Glu-Glu, γ-Glu-Val, and γ-Glu-Leu. However, these werefound as a result of separation with various columns, and the amounts ofthese peptides contained in the broth were not determined. In addition,there was not reported in all the above examples that γ-Glu-Abu wascontained.

GSH is biosynthesized by two kinds of enzymes called γ-glutamylcysteinesynthetase, which binds Glu and Cys to generate γ-Glu-Cys, andglutathione synthetase, which binds the generated γ-Glu-Cys and Gly togenerate GSH. The substrate specificities of the enzymes wereinvestigated in in vitro enzymatic reactions, and it was reported thatγ-Glu-Abu was generated from Glu and Abu as the substrates (Non-patentdocument 8). However, this report concerns an example using a bacterium,Proteus mirabilis, and does not concern investigation using yeast.Furthermore, although Abu can be used as a substrate in an in vitroenzymatic reaction, any Abu synthetic pathway is not known for yeasts.

Yeast extracts produced from yeast cells as a raw material areseasonings that have been widely used in the field of foodstuffs, andare highly accepted by consumers. Therefore, a yeast extract is morepreferred as a carrier of taste substances. Yeast strains containingminerals can be exemplified as the investigation concerning the use ofyeast as a carrier of taste substances. It is known that if a metal isadded to a medium, yeasts uptake the metal into the cells (Non-patentdocument 9). In particular, if trace elements such as zinc, iron,copper, manganese, selenium, molybdenum and chromium are added to themedium, yeasts can be used as a supply source of the desired enrichedelements as foodstuffs (Patent document 5). From this point of view,methods for producing mineral-containing yeast have been developed(Patent documents 6 to 8).

Furthermore, yeast incorporating such minerals may also enjoy a meritconcerning on taste. For example, there can be mentioned the yeastcontaining a large amount of magnesium (Patent document 9). Thisreference describes that although magnesium-enriched foodstuffscontaining inorganic magnesium salt were also marketed, a strongbitterness and astringency was noted due to the mineral salt. As aresult, it was quite more difficult to routinely eat themagnesium-enriched foodstuffs containing inorganic magnesium salt ascompared to foodstuffs containing naturally occurring magnesium. In thatcontext, Patent document 9 discloses a method to produce a foodstuffcontaining magnesium in natural form by letting yeast uptake magnesium.As for nutritional merit of yeast that uptakes minerals, the techniquedisclosed in Patent document 10 can be exemplified. According to thisreference, although zinc contributes to improvement of taste disorderand generative function, etc., zinc is still not taken in sufficientamounts. If zinc is added during the yeast cultivation process, yeastuptakes zinc into cells. In this case zinc is not accumulated in thecells as water-soluble form, but zinc is highly accumulated in the cellsas amorphous zinc form which binds with a protein or an amino acid. Whenthe amorphous zinc is taken into the human body, the amorphous zinc ismore efficiently absorbed into the body compared with crystalline zinc.As a result, absorption of zinc into the body can be improved by takingzinc-containing yeast, as compared to simply taking zinc itself.

As described above, a method comprised by making yeast uptake a targetsubstance and adding either the yeast or a yeast extract to foodstuffscan enjoy various advantages as compared to simply adding the targetsubstance itself to foodstuffs. However, unlike minerals, which areessential nutrients, the ability of yeast to uptake an amino acid or apeptide is strictly controlled, and simply applying the technique forincorporating minerals into yeast to the techniques for uptake of aminoacid or peptides was considered to be difficult.

As described above, although yeast cells or yeast extracts are preferredas a carrier of a γ-glutamyl compound such as γ-Glu-X as akokumi-imparting agent, there have been substantially no reports aboutyeast cells containing such a γ-glutamyl compound, and a method forproducing an extract prepared from the cells.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: WO2007/055393-   Patent document 2: WO2007/042288-   Patent document 3: WO2008/139945-   Patent document 4: WO2008/139946-   Patent document 5: Japanese Patent Laid-open (Kokai) No. 2004-298014-   Patent document 6: Japanese Patent Laid-open (Kokai) No. 54-157890-   Patent document 7: Japanese Patent Laid-open (Kokai) No. 60-75279-   Patent document 8: Japanese Patent Publication (Kokoku) No. 6-16702-   Patent document 9: Japanese Patent Laid-open (Kokai) No. 8-332081-   Patent document 10: Japanese Patent Laid-open (Kokai) No.    2008-099578

Non-Patent Documents

-   Non-patent document 1: Ueda et al., Agric. Biol. Chem., 54, 163-169    (1990)-   Non-patent document 2: Ueda et al., Biosci. Biotechnolo. Biochem.,    61, 1977-1980 (1997)-   Non-patent document 3: Wang et al., Journal of Biological Chemistry,    281, 8864-8870 (2006)-   Non-patent document 4: Gabriel et al., Biochemical and Biophysical    Research Communications, 378, 414-418 (2009)-   Non-patent document 5: Ohsu et al., Journal of Biological Chemistry,    285, 1016-1022 (2010)-   Non-patent document 6: Protein Nucleic acid Enzyme, 1988-7, VOL. 33,    NO. 9, ISSN 003909450, Special Issue “Epoch of glutathione    research”, pp. 1432-1433-   Non-patent document 7: Ronald et al., Journal of Biological    Chemistry, 240, p 2508-2511 (1965)-   Non-patent document 8: Nakayama et al., Agric. Biol. Chem., 45(12),    2839-2845 (1981)-   Non-patent document 9: B. Volesky, H. A., Appl. Microbiol.    Biotechnol., 42; 797-806 (1995)

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

An object of the present invention is to provide a yeast extract havinga kokumi-imparting effect of initial taste-imparting type, and a methodfor producing it.

Means for Achieving the Object

The inventors of the present invention previously found that γ-Glu-Abu(L-γ-glutamyl-L-γ-aminobutyric acid) had high CaSR agonist activity andextremely excellent kokumi-imparting effect, and in particular, it had ataste-imparting pattern of initial taste-imparting type. Then, theyfound that yeasts took up Abu (α-aminobutyric acid) or γ-Glu-Abu intocells thereof, and by preparing a yeast extract from yeast cultured in amedium containing Abu or γ-Glu-Abu, a yeast extract containing γ-Glu-Abucould be produced. Moreover, they also found that if theaminotransferase activity or/and the α-ketobutyric acid synthetaseactivity were increased, intracellular Abu production advanced. Further,they also found that by allowing γ-glutamyltransferase to act on a yeastextract raw material to which Abu had been added, a yeast extractcontaining γ-Glu-Abu could be produced. The present invention wasaccomplished on the basis of these findings.

The present invention thus relates to the followings.

(1) A yeast extract containing 0.2% or more of γ-Glu-Abu based on dryweight of the yeast extract.

(2) A yeast extract containing 0.5% or more of γ-Glu-Abu based on dryweight of the yeast extract.

(3) A yeast extract containing 1.0% or more of γ-Glu-Abu based on dryweight of the yeast extract.

(4) The yeast extract as mentioned above, wherein the yeast belongs tothe genus Saccharomyces or Candida.

(5) The yeast extract as mentioned above, which is obtained fromSaccharomyces cervisiae.

(6) The yeast extract as mentioned above, which is obtained from Candidautilis.

(7) A method for producing a yeast extract containing γ-Glu-Abu, whichcomprises culturing a yeast in a medium to which a compound selectedfrom Abu and γ-Glu-Abu is added, and preparing a yeast extract from theobtained cells.

(8) The method as mentioned above, wherein the compound is added to themedium in an amount of 10 ppm or more in the case of Abu, or 1 ppm ormore in the case of γ-Glu-Abu, and the yeast extract contains 0.2% ormore of γ-Glu-Abu based on dry weight of the yeast extract.

(9) The method as mentioned above, wherein the yeast belongs to thegenus Saccharomyces or Candida.

(10) The method as mentioned above, wherein the yeast is Saccharomycescervisiae.

(11) The method as mentioned above, wherein the yeast is Candida utilis.

(12) The method as mentioned above, wherein the yeast has one or both ofthe following characteristics:

(a) γ-glutamylcysteine synthetase activity is enhanced,

(b) glutathione synthetase activity is attenuated.

(13) A yeast having an increased γ-Glu-Abu content, which has beenmodified so that

activity of aminotransferase or/and activity of α-ketobutyric acidsynthetase are enhanced, and,

activity of γ-glutamylcysteine synthetase is enhanced, or/and activityof glutathione synthetase is attenuated.

(14) The yeast as mentioned above, wherein the aminotransferase is anenzyme encoded by the BAT1 gene.

(15) The yeast as mentioned above, wherein the aminotransferase is anenzyme encoded by the UGA1 gene.

(16) The yeast as mentioned above, wherein the α-ketobutyric acidsynthetase is an enzyme encoded by the CHA1 gene.

(17) The yeast as mentioned above, wherein a peptidase activity isfurther attenuated.

(18) A yeast extract produced from the yeast as mentioned above.

(19) A method for producing a yeast extract containing γ-Glu-Abu, whichcomprises allowing a γ-glutamyltransferase to act on a yeast extract rawmaterial to which Abu has beeb added.

(20) The method as mentioned above, wherein Abu is added in an amount of0.1% or more based on dry weight of the yeast extract raw material, andthe yeast extract contains 0.2% or more of Abu based on dry weight ofthe yeast extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mass chromatograms of Abu, γ-Glu-Abu, and γ-Glu-Abu-Glysamples.

FIG. 2 shows mass chromatograms of internal standard substances(3-methyl-His-d2, Gly-d2). In the mass chromatogram, 3MeHis-d2represents 3-methyl-His-d2.

FIG. 3 shows the construction procedure of the plasmid pCGSH1 containingthe GSH1 region of Candida utilis.

FIG. 4 shows the construction procedure of the vector pCGSH1-URA3 forexpression of GSH1 having the URA3 gene.

FIG. 5 shows the relation between added Abu concentration and γ-Glu-Abuproduction amount in the yeast extract on which γ-glutamyltransferasewas made to act.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in detail.

The yeast extract of the present invention is a yeast extract containing0.2% or more of γ-Glu-Abu based on dry weight of the yeast extract.

The yeast extract of the present invention contains γ-Glu-Abu in anamount of 0.2% or more, preferably 0.5% or more, more preferably 1.0% ormore, particularly preferably 2.0% or more, based on dry weight of theyeast extract.

The yeast used as the raw material of the yeast extract of the presentinvention is the same as the yeast used for the method of the presentinvention described later.

The form of the yeast extract of the present invention is notparticularly limited, and it may be in the form of powder or solution.The yeast extract can have the same uses as that of conventional yeastextracts, for example, seasonings, food additives, health foods, and soforth. The yeast extract of the present invention is excellent in itskokumi-imparting effect. Since the kokumi-imparting effect is morestrongly reinforced in the presence of umami or salty taste, an umamisubstance such as sodium L-glutamate and tasty nucleotides, and/or asalty substance such as sodium chloride may be added to the yeastextract. Further, since the yeast extract of the present invention hasespecially a superior effect of imparting kokumi as the initial taste,it may be mixed with a kokumi-imparting substance showing a differentkokumi-imparting pattern, such as GSH or γ-Glu-Val-Gly, or a yeastextract containing such a substance. Moreover, an umami substance and/ora salty substance may be added to seasonings, food additives or healthfoods together with the yeast extract of the present invention. Thetastes are classified into initial taste, middle taste, and aftertaste.Although these are relative concepts, they are usually defined as tastessensed in the periods of 0 to 2 seconds, 2 to 5 seconds, and after 5seconds after eating, respectively. The “initial-middle taste” mentionedabove is a taste sensed in the period of 0 to 5 seconds after eating,and the “middle-after taste” mentioned later is a taste sensed in theperiod of 2 second to about 30 seconds after eating.

The yeast extract of the present invention can be produced by, forexample, the method of the present invention described below.

The first method of the present invention is a method for producing ayeast extract containing γ-Glu-Abu, which includes the steps ofculturing a yeast in a medium to which a compound selected from Abu andγ-Glu-Abu is added, and preparing a yeast extract from the obtainedcells.

Any yeast from any wild-type strains, or various mutant strains orrecombinant strains can be used for this invention, so long as thechosen yeast can intracellularly uptake Abu and/or γ-Glu-Abu andaccumulate γ-Glu-Abu in the cells. Examples of the mutant strains orrecombinant strains include a strain with enhanced activity ofγ-glutamylcysteine synthetase (GSH1), a strain with attenuated activityof glutathione synthetase (GSH2), and a strain having two of theforegoing characteristics. Examples of the mutant strains or recombinantstrains also include a strain with attenuated activity of a peptidasethat decompose intracellular peptides, for example, an enzyme encoded bythe DUG1 gene, DUG2 gene, DUGS gene, or ECM38 gene. Further, in additionto the enhanced activity of GSH1 and/or attenuated activity of GSH2, apeptidase activity of the yeast may be attenuated. The nucleotidesequences of the aforementioned genes are disclosed in the SaccharomycesGenome Database (http://www.yeastgenome.org/).

The yeast is not particularly limited, so long as the chosen yeast canaccumulate γ-Glu-Abu in the cells thereof. Examples include yeastsbelonging to the genus Saccharomyces such as Saccharomyces cerevisiae,those belonging to the genus Candida such as Candida utilis, thosebelonging to the genus Pichia such as Pichia pastoris, and thosebelonging to the genus Schizosaccharomyces such as Schizosaccharomycespombe. Among these, Saccharomyces cerevisiae and Candida utilis arepreferred, which are frequently used for production of yeast extracts.The yeast may be a monoploid, or may have diploidy or a further higherpolyploidy.

Methods for enhancing activity of an enzyme or protein such as GSH1include a method of enhancing expression of a gene coding for it.Methods for enhancing expression of a gene include a method of replacingthe promoter of the gene on the chromosome with a stronger promoter, amethod of inserting the target gene into the chromosome to increase copynumber thereof, a method of incorporating a plasmid containing thetarget gene into the yeast, a method of activating a transcriptionfactor of the gene coding for the target enzyme, and so forth.

As the promoter, a highly active conventional promoter may be obtainedby using various reporter genes, or a known high expression promotersuch as ADH1, PGK1, PDC1, TDH3, TEF1 and HXT7 may be used.Alternatively, for increasing copy number of a target gene, for example,a plasmid having the replication origin of CEN4, or a multi-copy plasmidhaving the replication origin of 2 μm DNA, to which a target gene isinserted, may be used. Furthermore, a transposon may be used in order tointroduce a target gene into an arbitrary region of the chromosome, orthe target gene may be introduced by using rDNA sequences as a target,which is present in a copy number of 150 in the cell.

Methods for attenuating activity of an enzyme or protein such as GSH2and peptidase include a method of replacing the promoter of the genecoding for any of these on the chromosome with a weaker promoter toattenuate the expression, a method of introducing a mutation into thetarget enzyme to reduce the activity thereof, a method of deleting apart of or the entire gene of the target enzyme from the chromosome, amethod of inactivating the target enzyme gene by inserting anothersequence into the gene, and so forth.

The attenuated enzyme activity include the activity lower than that of awild-type strain and complete deficiency of the enzyme activity.

Enhancement of the activity of γ-glutamylcysteine synthetase isdisclosed in, for example, U.S. Pat. No. 7,553,638; Otake Y. et al.,Bioscience and Industry, volume 50, No. 10, pp. 989-994, 1992, and soforth. Although disruption of the glutathione synthetase gene isdisclosed in U.S. Pat. No. 7,553,638, the glutathione synthetaseactivity can also be reduced by inactivating YAP1, which is atranscription factor of the gene coding for γ-glutamylcysteinesynthetase.

The nucleotide sequences of the genes coding for GSH1, GSH2, and YAP1 ofSaccharomyces cerevisiae are disclosed in Saccharomyces Genome Database(http://www.yeastgenome.org/). The nucleotide sequences of the genescoding for GSH1 and GSH2 of Candida utilis are disclosed in U.S. Pat.No. 7,553,638. The nucleotide sequence of the gene coding for YAP1 ofCandida utilis is disclosed in Japanese Patent Laid-open (Kokai) No.2006-75122.

Such a yeast as mentioned above may be a yeast obtained by screeningfrom nature, breeding based on mutagenesis, or breeding based on geneticengineering. For the mutagenesis, various agents such as EMS, DAPA, andNTG can be used, and an objective mutant strain can be isolated byspreading yeast cells subjected to a mutation treatment on an optimalmedium, and choosing a strain of which GSH1 activity is enhanced or astrain of which GSH2 activity is attenuated from the grown strains.

The method for breeding by genetic engineering is not particularlylimited, and conventional methods can be used. In particular, geneticengineering methods for Saccharomyces cervisiae are specificallydescribed in many books. Moreover, various kinds of methods have beenreported also for Candida utilis in recent years, and they may be used.Specific methods are described in such prior references as, for example,Norihiko Misawa, Chemical Engineering, June, 1999, pp. 23-28; LuisRodriguez et al., FEMS Microbiology Letters, 165, 335-340 (1998);WO98/07873; Japanese Patent Laid-open (Kokai) No. 8-173170; WO95/32289;Keiji Kondo et al., Journal of Bacteriology, December 1995, Vol. 177,No. 24, pp. 7171-7177; WO98/14600; Japanese Patent Laid-open (Kokai)Nos. 2006-75122, 2006-75123, 2007-089441, and 2006-101867, and these canbe appropriately referred to.

The method for producing the yeast extract is explained below.

First, a yeast is cultured in a medium. The medium is not particularlylimited, so long as a medium in which the yeast can proliferate ischosen, and is not limited to the SD medium described in the examples. Amedium usually used for industrial purposes can be used. Examples of themedium include, for example, media containing glucose, sucrose,molasses, ethanol, acetic acid, spent sulfite liquor, or the like as acarbon source, urea, ammonia, ammonium sulfate, ammonium chloride,nitrate, or the like, or corn steep liquor, casein, yeast extract,peptone, soybean protein decomposition product, or the like as anitrogen source, phosphoric acid, potassium phosphate, ammoniumphosphate, superphosphate of lime, potassium chloride, potassiumhydroxide, magnesium sulfate, magnesium chloride, and so forth asphosphoric acid, potassium, and magnesium sources, and an appropriatecombination of mineral salts of copper, manganese, zinc, iron ion, etc.as trace elements.

When the yeast is cultured, Abu, γ-Glu-Abu, or both of these is/areadded to the aforementioned medium. Abu represents α-aminobutyric acid,and Glu represents glutamic acid. Abu and Glu are L-forms. Thesecompounds may be present in the medium from the start of the culture, ormay be added to the medium at an arbitrary time during the culture. Whenthe compounds are added to the medium during the culture, they can bepreferably added at 0 to 50 hours before the end of the culture (0 hourmeans that the culture is terminated immediately after the addition),more preferably 0.1 to 24 hours before the end of the culture,particularly preferably 0.5 to 6 hours before the end of the culture.Furthermore, when the peptides are added during the culture, they may becontinuously added.

Abu and/or γ-Glu-Abu to be added to the medium may be a purified product(pure substance), or may be a composition containing such a compound orcompounds, so long as the composition contains required amounts of thesecompounds.

Prior to the culture in the medium containing the compound(s), apreculture may be performed. The medium used for the preculture may ormay not contain the compound(s).

When the compound is added to the medium at the start of the culture,Abu is added in an amount of usually 10 ppm or more, preferably 25 ppmor more, more preferably 50 ppm or more, further preferably 100 ppm, andγ-Glu-Abu is added in an amount of usually 1 ppm or more, preferably 5ppm or more, more preferably 10 ppm or more, in terms of the finalconcentration in the culture broth at the time of the addition. Whenboth Abu and γ-Glu-Abu are added, concentrations thereof can bedetermined to be within the aforementioned ranges. Although the maximumamount of the compound is not particularly limited, it is, for example,100,000 ppm or less from an aspect of production cost, and it is usually10,000 ppm or less, preferably 1,000 ppm or less, more preferably 500ppm or less. When the compound is added during an arbitrary period inthe middle of the culture, or it is continuously added, it may be addedin a total amount equivalent to an amount that can provide the finalconcentration mentioned above when the compound is added in such anamount at the time of the start of the culture.

As the culture conditions, the same conditions as those used for usualproduction of yeast extracts can be used, and they may be suitablychanged according to the chosen yeast. Arbitrary methods such as batchculture, fed-batch culture, and continuous culture may be used. When theyeast is Saccharomyces cerevisiae or Candida utilis, it is preferablyaerobically cultured by shaking or the like at 25 to 35° C., morepreferably 27 to 33° C., still more preferably 28 to 32° C.

If the yeast is cultured as described above, γ-Glu-Abu accumulates inthe cells of the yeast. When Abu is added to the medium, Abu accumulatesin the cells, and in addition, γ-Glu-Abu also accumulates. This isbecause Abu taken up into the cells is converted into γ-Glu-Abu by theaction of intracellular γ-glutamylcysteine synthetase as shown in theexample section mentioned later. As shown in the example section, thecontent of γ-Glu-Abu or γ-Glu-Abu-Gly in the yeast did not correlatewith the content of GSH in the cells, and therefore it is consideredthat yeast extracts produced by the conventional methods do not containγ-Glu-Abu at a high concentration, even if they are produced from ayeast containing GSH at a high concentration. It is estimated that thisis because generation of Abu is limited in the cells as described in theexample section mentioned later. According to a preferred embodiment,the yeast cultured as mentioned above contains γ-Glu-Abu in an amount of0.04% or more, preferably 0.1% or more, more preferably 0.15% or more,still more preferably 0.2% or more, particularly preferably 0.4% ormore, based on dry weight of the cells.

The yeast extract can be prepared from the obtained yeast in the samemanner as that used for conventional production of yeast extracts. Theyeast extract may be obtained by subjecting the yeast cells to hot waterextraction and processing the extract, or by digesting the yeast cellsby self-digestion or with an enzyme and processing the digestionproduct. Furthermore, the obtained yeast extract may be concentrated,may be in the form of paste, or may be dried and thereby made intopowdered form, as required.

In such a manner as described above, a yeast extract containing anincreased amount of γ-Glu-Abu can be obtained. According to a preferredembodiment, the yeast extract contains γ-Glu-Abu in an amount of 0.2% ormore, more preferably 0.5% or more, still more preferably 1.0% or more,particularly preferably 2.0% or more, based on dry weight of the yeastextract.

The second method of the present invention will be explained. The secondmethod relates to a yeast of which Abu synthesis ability is enhancedwithin the yeast cell. Although Abu synthetic pathway in a yeast cellhad not conventionally been known, it was found that it was producedfrom α-ketobutyric acid with aminotransferase in yeast as shown in theexample section. Therefore, if Abu synthesis ability is enhanced in ayeast cell, the γ-Glu-Abu accumulation ability is improved. The Abusynthesis ability can be enhanced by enhancing the aminotransferaseactivity or the α-ketobutyric acid synthetase activity. The activity ofaminotransferase or α-ketobutyric acid synthetase can be enhanced in thesame manner as that used for GSH1 and so forth.

One embodiment of the yeast of the present invention is a yeast of whichaminotransferase activity is enhanced. Another embodiment of the yeastof the present invention is a yeast of which α-ketobutyric acidsynthetase activity is enhanced. Still another embodiment of the yeastof the present invention is a yeast both of which aminotransferaseactivity and α-ketobutyric acid synthetase activity are enhanced. Insuch a yeast of which aminotransferase activity or/and α-ketobutyricacid synthetase activity are enhanced, the activity ofγ-glutamylcysteine synthetase (GSH1) may be enhanced, or the activity ofglutathione synthetase (GSH2) may be attenuated as in the first methodof the present invention. Alternatively, the yeast may have two of thesecharacteristics. Furthermore, a peptidase that decomposes intracellularpeptides, for example, an enzyme encoded by the DUG1 gene, DUG2 gene,DUG3 gene, or ECM38 gene, may be attenuated.

A yeast of which Abu synthesis ability is enhanced, for example, a yeastmodified so that the γ-glutamylcysteine synthetase activity and theaminotransferase activity, or/and the α-ketobutyric acid synthetaseactivity are enhanced accumulates a marked amount of γ-Glu-Abu, evenwhen it is cultured in a medium to which Abu and γ-Glu-Abu are notadded. The yeast of a preferred embodiment, for example, such a yeast ofwhich activity of aminotransferase encoded by the BAT1 mentioned below,and the GSH1 activity are enhanced, and of which GSH2 activity isattenuated, preferably contains γ-Glu-Abu in an amount of 0.04% or more,more preferably 0.1% or more, still more preferably 0.15% or more,further preferably 0.2% or more, particularly preferably 0.4% or more,based on dry weight of the cells. A yeast extract prepared from such ayeast contains 0.2% or more of γ-Glu-Abu based on dry weight of theyeast extract.

According to another embodiment, for example, the aforementioned yeastof which activity of serine (threonine) deaminase encoded by CHA1 isfurther enhanced preferably contains γ-Glu-Abu in an amount of 0.1% ormore, more preferably 0.15% or more, still more preferably 0.2% or more,further preferably 0.4% or more, particularly preferably 0.5% or more,based on dry weight of the cells. When a yeast of which Abu synthesisability is enhanced is cultured, Abu and/or γ-Glu-Abu may be added tothe medium.

The yeast is not particularly limited, so long as the chosen yeast canaccumulate γ-Glu-Abu in the cells thereof. Examples include yeastsbelonging to the genus Saccharomyces such as Saccharomyces cerevisiae,those belonging to the genus Candida such as Candida utilis, thosebelonging to the genus Pichia such as Pichia pastoris, and thosebelonging to the genus Schizosaccharomyces such as Schizosaccharomycespombe. Among these, Saccharomyces cerevisiae and Candida utilis arepreferred, which are frequently used for production of yeast extracts.The yeast may be a monoploid, or may have diploidy or a further higherpolyploidy.

Examples of aminotransferase of yeast include alanine: glyoxylateaminotransferase, branched-chain amino acid transaminase, aspartateaminotransferase, γ-aminobutyrate transaminase, and so forth. The genescoding for these enzymes in Saccharomyces cervisiae have already beenspecified, and they are encoded by AGX1 (systematic name: YFL030W), BAT1(systematic name: YHR208W), BAT2 (systematic name: YJR148W), AAT1(systematic name: YKL106W), AAT2 (systematic name: YLR027C), and UGA1(systematic name: YGR019W), respectively. Moreover, various genes havebeen also reported for Candida utilis in recent years, and homologuegenes thereof can also be easily specified by identifying the totalgenome sequence using recent advanced sequencers, and used. Among these,BAT1 and UGA1 are preferred, and BAT1 is particularly preferred, sinceit shows a marked effect as described in the example section describedlater. Although activity of one kind of aminotransferase may beenhanced, activities of arbitrary two or more kinds of aminotransferasesmay also be enhanced.

Examples of the α-ketobutyric acid synthetase of yeast include theserine (threonine) deaminase encoded by the CHA1 gene (systematic name:YCL064C), and the threonine deaminase encoded by the ILV1 gene(systematic name: YER086W). Although activity of one kind ofα-ketobutyric acid synthetase may be enhanced, activities of arbitrarytwo or more kinds of α-ketobutyric acid synthetases may also beenhanced.

The activities of aminotransferase and α-ketobutyric acid synthetase canbe enhanced by enhancing expression of genes coding for the enzymes asin the case of enhancement of the GSH1 activity.

Production of a yeast extract using a yeast having enhanced activity ofaminotransferase or/and enhanced activity of α-ketobutyric acidsynthetase, and containing γ-Glu-Abu can be performed in the same manneras that explained for the first method.

The third method of the present invention is a method for producing ayeast extract containing γ-Glu-Abu, which includes the step of allowinga γ-glutamyltransferase to act on a yeast extract raw material to whichAbu has been added.

If a γ-glutamyltransferase is allowed to act on Abu, γ-Glu-Abu isgenerated. Therefore, a yeast extract containing γ-Glu-Abu can also beobtained by allowing a γ-glutamyltransferase to act on a yeast extractcontaining Abu. The yeast extract containing Abu may be prepared from ayeast cultured in a medium containing Abu, or may be obtained by addingAbu to a yeast extract raw material.

As the yeast extract raw material, a yeast extract obtained by aconventional method can be used.

Abu is usually added to the yeast extract in an amount of 0.1% or more,preferably 1% or more, more preferably 5% or more, still more preferably10% or more, based on dry weight of the yeast extract raw material.

The reaction catalyzed by the γ-glutamyltransferase is performed in anaqueous solvent such as water or buffers. Specifically, for example, theyeast extract raw material is dissolved in the aqueous solvent, and theγ-glutamyltransferase is added. The reaction conditions can be suitablydetermined according to the γ-glutamyltransferase to be used. Thereaction is usually allowed at pH 3 to 9 and 15 to 70° C. for 1 to 300minutes, preferably at pH 5 to 8 and 30 to 70° C. for 5 to 150 minutes.

Concentration of the yeast extract raw material in the aqueous solventmay be determined in view of ease of handling. The concentration isusually 0.1 to 50%, preferably 0.5 to 20%, in terms of dry weight of theyeast extract raw material.

Examples of the γ-glutamyltransferase include glutaminase, γ-glutamyltranspeptidase (γ-GTP), and so forth. As for the amount of the enzyme,in the case of γ-GTP, it is usually 0.001 to 1000 units/ml, preferably0.005 to 100 units/ml, more preferably 0.01 to 25 units/ml, mostpreferably 0.05 to 10 units/ml, wherein 1 unit is defined to be theactivity of liberating 1.0 μmole of p-nitroaniline fromγ-glutamyl-p-nitroanilide per 1 minute in a solution at pH 8.5 and 25°C. (definition described in Sigma General Catalogue, 2008-2009 Edition,p. 917). The amount of glutaminase can also be determined in a mannersimilar to that for γ-GTP.

After the enzymatic reaction, a treatment for inactivating theγ-glutamyltransferase, for example, a heat treatment at 80 to 100° C.,may be performed, or may not be performed.

As a substrate of the γ-glutamyltransferase, a γ-glutamyl compound, forexample, GSH, may be added to the reaction mixture. GSH contained in theyeast extract may also be used as a substrate. In this case, a yeastextract prepared from a yeast in which the content of GSH is increased,for example, a yeast in which activities or activity of GSH1 and/or GSH2is enhanced, can be used. Although a greater GSH content in the yeastextract is more preferred, it is usually 1 to 50%, preferably 1 to 30%,more preferably 5 to 20%, based on dry weight of the yeast extract.Alternatively, glutamine can also be used like GSH.

In such a manner as described above, a yeast extract in which the amountof γ-Glu-Abu is increased is obtained. According to a preferredembodiment, the yeast extract contains γ-Glu-Abu in an amount of 0.02%or more, more preferably 0.5% or more, still more preferably 1.0% ormore, particularly preferably 2.0% or more, based on dry weight of theyeast extract.

The obtained yeast extract may be concentrated, or may be in the form ofpaste, or may be dried and thereby made into powdered form, as required.

Another kokumi substance may be added to a yeast extract obtained by anyof the aforementioned first to third methods of the present invention.Examples of such a kokumi substance include, for example, a peptide suchas γ-Glu-X and γ-Glu-X-Gly (X represents an amino acid or an amino acidderivative), specifically GSH and γ-Glu-Val-Gly, and a yeast extractcontaining these. As described above, a yeast extract that has kokumifor a broad range from the initial taste to aftertaste can be produced.In the case of GSH, in particular, in view of the balance of kokumi ofthe initial taste and the aftertaste, the ratio of GSH with respect toγ-Glu-Abu is preferably 0.3 or more, more preferably 0.5 or more, stillmore preferably 1.0 or more, particularly preferably 3.0 or more.

EXAMPLES

Hereafter, the present invention will be more specifically explainedwith reference to examples.

However, the present invention is no way limited by the followingexamples. Unless specially mentioned, the amino acids and amino acidderivatives mentioned in the examples are L-forms.

Reference Example 1 Evaluation of Kokumi-Imparting Activity of γ-Glu-Abu

Degree of kokumi-imparting activity of γ-Glu-Abu was investigatedthrough a quantitative organoleptic evaluation test.

The quantitative organoleptic evaluation test was carried out asfollows. Strength of the kokumi-imparting activity of a test compoundwas measured by using a mixture containing the test compound at 0.001 to0.5 g/dl in distilled water containing sodium glutamate (0.05 g/dl),inosine monophosphate (0.05 g/dl), and sodium chloride (0.5 g/dl). Asample of the distilled water containing sodium glutamate, inosinemonophosphate, and sodium chloride to which any test compound was notadded was used as a no addition control. Samples that became acidicrelative to the no addition control after dissolution of the testcompound were used after pH of them was adjusted with NaOH to be withinthe range of ±0.2 from that of the no addition control.

The test was performed with organoleptic scores of 0 for the control, 3for strong effect, and 5 for extremely strong effect, and n=4. Moreover,in order to make the criteria more definite, the scores of 0.001 g/dl ofγ-Glu-Val-Gly for the initial taste and the middle-after taste weredefined to be 3.0. The “middle-after taste” is a taste sensed in theperiods for the middle taste and the aftertaste. Specifically, theinitial taste, middle taste, and aftertaste are tastes sensed in theperiods of 0 to 2 seconds, 2 to 5 seconds, and after 5 seconds, aftereating, respectively, and the “middle-after taste” is a taste sensed inthe period from 2 seconds to around 30 seconds. For the scoring, thelinear scale method was used, in which determined scores were noted on astraight line on which positions of scores of from −5 to 5 wereindicated. The panelists were consisted of persons who had experience ofdevelopment of seasonings for foodstuffs for one year or longer intotal, and could judge the difference of the titers of γ-Glu-Cys-Gly andγ-Glu-Val-Gly added to the tasty and salty solution to be about 10 times(this ability was periodically confirmed). Although γ-Glu-Abu showedkokumi-imparting activity in a broad range within the aforementionedaddition concentration range, the result for a typical concentration isshown in Table 1.

The result of similar evaluation for γ-Glu-Ala is also shown in Table 2.The both are initial taste-imparting type substances giving high scoresfor the initial taste, but it was found that γ-Glu-Abu was a dipeptideshowing an extremely high titer.

TABLE 1 Intensity of kokumi Middle- Organoleptic Concentration Initialafter evaluation Compound (g/dl) taste taste profile γ-Glu-Abu 0.005 3.83.2 Atsumi (total taste and harmony) was enhanced from initial taste.γ-Glu-Ala 0.2 4.5 4.3 Tastes belonging to acidic and sweet tastes wereenhanced from initial taste.

Kokumi-imparting activities of γ-Glu-Cys and other dipeptides weredetermined by the same quantitative organoleptic evaluation test asmentioned above. The results are shown in Table 2.

TABLE 2 Intensity of kokumi Middle- Organoleptic Concentration Initialafter evaluation Compound (g/dl) taste taste profile γ-Glu-Cys 0.01 3.13.1 Middle-after taste was mainly enhanced, and slight sulfur smell wassensed. γ-Glu-Ser 0.2 3.6 3.0 Strong initial taste was obtained, butstrange taste was sensed. γ-Glu-Val 0.01 3.1 2.4 Aftertaste wasextremely weak.

It was found that γ-Glu-Abu had superior kokumi-imparting activity, andshowed marked rise of the initial taste in the taste pattern. This riseof the initial taste is one of the extremely advantageouscharacteristics of γ-Glu-Abu compared with γ-Glu-Cys. Moreover,γ-Glu-Abu shows superior storage stability, and this is also one of theadvantageous characteristics compared with γ-Glu-Cys. Further, since thenumber of residues contained in γ-Glu-Abu is as small as two, it can bemore easily produced at lower cost compared with tripeptides containingthree amino acid residues, and this is industrially extremelyadvantageous.

Example 1 Detection of Abu, γ-Glu-Abu and γ-Glu-Abu-Gly in Various YeastExtracts

Abu, γ-Glu-Abu and γ-Glu-Abu-Gly contents in various yeast extracts weremeasured by fluorescence derivatization of the compounds with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), and detection byLC-MS/MS according to the method described below. Specifically, to 2.5μL of a sample diluted to an appropriate concentration, or 2.5 μL eachof standard solutions containing 1 μM Abu, γ-Glu-Abu or γ-Glu-Abu-Gly,2.5 μL of Milli-Q water, 5 μL of a 5 μM internal standard substancesolution (3-methyl-His-d2 (Sigma) or Gly-d2 (Sigma), both are labeledwith stable isotope), and 30 μL of a borate buffer (attached toAccQ-Fluor (registered trademark) Reagent Kit, Nihon Waters) were added.To each mixture, 10 μL of an AQC reagent solution (prepared bydissolving the reagent powder of the aforementioned reagent kit in 1 mLof acetonitrile) was added. This mixture was heated at 55° C. for 10minutes, and then 100 μL of 0.1% formic acid aqueous solution was addedto the mixture to prepare a sample for analysis.

Then, the sample for analysis prepared as described above was subjectedto separation by the reverse phase liquid chromatography describedbelow, and then introduced into a mass spectrometer. The separationconditions were as follows.

(1) HPLC: Agilent 1200 Series

(2) Separation column: Unison UK-Phenyl; internal diameter, 2.0 mm;length, 100 mm; particle size, 3 μm (Imtakt)(3) Column oven temperature: 40° C.(4) Mobile phase A: 25 mM Ammonium formate (pH 6.0, adjusted withaqueous ammonia)(5) Mobile phase B: methanol(6) Flow rate: 0.25 mL/min(7) Elution conditions: Elution was performed by using mixtures of themobile phase A and the mobile phase B. The ratios of the mobile phase Bto the mixtures are as follows: 0 minute (5%), 0 to 17 minutes (5 to40%), 17 to 17.1 minutes (40 to 80%), 17.1 to 19 minutes (80%), 19 to19.1 minutes (80 to 5%), 19.1 to 27 minutes (5%).

Then, derivatized compounds of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly elutedunder the aforementioned separation conditions were introduced into amass analyzer, and quantified by mass chromatography. The analysisconditions were as follows.

(1) Mass analyzer: AB Sciex API3200 QTRAP(2) Detection mode: Selected Ion Monitoring (positive ion mode)

(3) Selected ion: Table 3

TABLE 3 First mass Second mass Derivatized analyzer analyzer compound(Q1) (Q3) Abu 274.2 171.1 γ-Glu-Abu 403.4 171.1 γ-Glu-Abu-Gly 460.4171.1 3-methyl-His-d2 343.4 171.1 Gly-d2 248 171.1

The derivatized compounds of Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly werequantified by using analysis software, Analyst ver. 1.4.2 (AB Sciex). Asthe internal standard substance for performing the quantification, aderivatized compound of 3-methyl-His-d2 was used in the case of thederivatized compound of Abu, and a derivatized compound of Gly-d2 wasused in the case of the derivatized compounds of γ-Glu-Abu andγ-Glu-Abu-Gly. The analysis results (mass chromatograms) of Abu,γ-Glu-Abu, and γ-Glu-Abu-Gly as well as the derivatized internalstandard amino acid are shown in FIGS. 1 and 2. It was found that, byusing this method, Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly in various samplescould be measured.

At the time of the quantification of γ-Glu-Abu, a contaminated peak wasvery rarely observed, and in such a case, the quantification wasperformed by using selected ion of 145.2 or 104.1 for the second massanalyzer.

Example 2 Measurement of γ-Glu-Abu Content in Various CommerciallyAvailable Yeast Extracts

γ-Glu-Abu contents in various commercially available yeast extracts(based on dry weight of the yeast extracts) were measured by using themethod of Example 1. GSH contents were also measured in a conventionalmanner. The results are shown in Table 4.

TABLE 4 γ-Glu-Abu γ-Glu-Abu-Gly GSH γ-Glu-Abu/GSH Brand A 109 ppm 348ppm  1461 ppm 0.075 Brand B  15 ppm  50 ppm  186 ppm 0.080 Brand C  95ppm 440 ppm  874 ppm 0.108 Brand D 424 ppm 249 ppm 11478 ppm 0.037 BrandE 920 ppm 316 ppm 66521 ppm 0.014

As shown in Table 4, the γ-Glu-Abu contents in various yeast extractswere in the range of 15 to 920 ppm. Further, the γ-Glu-Abu/GSH ratio wasnot constant, and in particular, there was observed a tendency that theratio was more decreased in a yeast extract having a higher GSH content.It is considered that this is because GSH1 and GSH2 responsible for theGSH biosynthetic pathway can recognize Abu and γ-Glu-Abu as a substrate,but first of all, the intracellular generation amount of Abu is limited,and even if the generation pathway of GSH is enhanced, a large amount ofγ-Glu-Abu cannot be accumulated due to lack of Abu, as shown in thefollowing examples. This suggests that the γ-Glu-Abu content in theknown high GSH content yeasts is not so high.

Example 3 Effect of Addition of Abu to Saccharomyces cervisiae TypeStrain, S288C Strain

Then, γ-Glu-Abu content in the S288C strain, which is a type strain ofSaccharomyces cervisiae, was measured. Moreover, effect of addition ofthe precursor, Abu, to the medium was also investigated. The S288Cstrain is deposited at the independent administrative agency, NationalInstitute of Technology and Evaluation, Biological Resource Center(NBRC, NITE Biological Resource Center, 2-5-8 Kazusakamatari,Kisarazu-shi, Chiba-ken, 292-0818, Japan) with the number of NBRC 1136,and can be provided therefrom. This strain is also deposited at theAmerican Type Culture Collection (12301 Parklawn Drive, Rockville, Md.20852, United States of America) with the number of ATCC 26108, and canbe provided therefrom.

One loop of the S288C strain was inoculated into the SD medium (50 ml in500 ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hourswith shaking at a velocity of 120 rpm.

Composition of SD Medium:

Glucose 2% Nitrogen Base 1-fold concentration(Nitrogen Base of 10-fold concentration was obtained by dissolving amixture of 1.7 g of Bacto Yeast Nitrogen Base w/o Amino Acids andAmmonium Sulfate (Difco) and 5 g of ammonium sulfate in 100 ml ofsterilized water, adjusting the solution to about pH 5.2, andsterilizing the solution by filter filtration.)

Absorbance of the obtained culture broth was measured, the culture brothwas inoculated into the SD medium or the SD medium containing 10, 50 or100 ppm of Abu as the final concentration (400 ml in a 2 L-volumeconical flask with baffle fins), so that OD660 was 0.01 at the start ofthe culture (absorbance was measured by using DU640 SPECTROPHTOMETER,BECKMAN COULTER), and the yeast cells were cultivated at 30° C. for 19hours with shaking by rotation at a velocity of 120 rpm. From theobtained culture broth, 400 OD units of the cells (1 OD unit is definedas cells contained in 1 ml of culture broth of which OD660 is 1) werecollected by centrifugal separation. The supernatant was removed as muchas possible, and the residual cells were suspended in 45 ml of Milli-Qwater. The cells were collected again by centrifugal separation, andresuspended in 45 ml of Milli-Q water. By repeating this operation 3times in total, the medium was completely removed from the cells. Thewashed cells were suspended in about 1.5 ml of Milli-Q water, and thesuspension was heated at 70° C. for 10 minutes. By this step, theextractable components contained in the cells were extracted. Then, theextract and the cell residue were separated by centrifugation.

Cell debris were removed from the extract using a centrifugal filtrationmembrane of 10 kDa cutoff (Amicon Ultra—0.5 mL 10K, MILLIPORE, CatalogueNumber UFC501096)), the obtained fraction was derivatized with the AQCreagent in the same manner as that used in Example 1, and Abu,γ-Glu-Abu, and γ-Glu-Abu-Gly were measured by LC-MS/MS. Furthermore, drycell weight was measured after the washed cells were dried at 104° C.for 4 hours. From the amounts of Abu, γ-Glu-Abu, and γ-Glu-Abu-Glycontained in a certain volume of culture broth and dry cell weightmeasured as described above, the contents of Abu, γ-Glu-Abu, andγ-Glu-Abu-Gly based on weight of dried cells were calculated. Theresults are shown in Table 5.

TABLE 5 Abu γ-Glu-Abu γ-Glu-Abu-Gly (ppm) (ppm) (ppm) SD medium 2 22 28Medium containing Abu (10 ppm) 33 170 51 Medium containing Abu (50 ppm)564 1025 155 Medium containing Abu (100 ppm) 450 1828 615

As shown in Table 5, it was found that, by adding Abu to the medium, theγ-Glu-Abu content in the cells was increased. Moreover, since not onlyγ-Glu-Abu but also γ-Glu-Abu-Gly accumulated, it was found that a partof γ-Glu-Abu was converted into γ-Glu-Abu-Gly by a certain enzymaticreaction using γ-Glu-Abu as a substrate in the yeast cells.

In addition, Abu in the washing solution obtained in the cell washingstep was measured, but Abu was not contained in the final washingsolution. The cell washing operation was performed further once again,and therefore it was confirmed that Abu in the medium was fully removedby the four-step separation in the washing step, and was not carriedover into the cell extract. Further, γ-Glu-Abu was not detected in thefinal washing solution also in the γ-Glu-Abu addition experimentsdescribed in the examples mentioned below.

Example 4 Effect of Addition of γ-Glu-Abu to S288C Strain (1)

Then, effect of addition of γ-Glu-Abu to the medium at the time ofculturing the S288C strain was investigated. One loop of the S288Cstrain was inoculated into the SD medium (50 ml in a 500 ml-volumeSakaguchi flask), and cultivated at 30° C. for 24 hours with shaking ata velocity of 120 rpm. Absorbance of the obtained culture broth wasmeasured (absorbance was measured by using DU640 SPECTROPHTOMETER,BECKMAN COULTER), the culture broth was inoculated into the SD medium orthe SD medium containing 10 or 100 ppm of γ-Glu-Abu as the finalconcentration (400 ml in a 2 L-volume conical flask with baffle fins),so that OD660 was 0.01 at the start of the culture, and the yeast cellswere cultivated at 30° C. for 19 hours with shaking by rotation at avelocity of 120 rpm. Extract was obtained from the obtained culturebroth in the same manner as that of Example 3, and contents of thecompounds in the cells were measured.

As shown in Table 6, it was found that when the S288C strain wascultured in a medium containing γ-Glu-Abu, the S288C strain took upγ-Glu-Abu in the medium and accumulated it in the cells. Moreover, notonly γ-Glu-Abu, but also γ-Glu-Abu-Gly was accumulated, as in Example 3.

TABLE 6 γ-Glu-Abu γ-Glu-Abu-Gly (ppm) (ppm) SD medium 21 39 Mediumcontaining γ-Glu-Abu (10 ppm) 1796 657 Medium containing γ-Glu-Abu (100ppm) 23338 2986

Example 5 Effect of Addition of γ-Glu-Abu to S288C Strain (2)

Then, effect of delayed addition of γ-Glu-Abu to the medium at the timeof culturing the S288C strain was investigated. One loop of the S288Cstrain was inoculated into the SD medium (50 ml in a 500 ml-volumeSakaguchi flask), and cultivated at 30° C. for 24 hours with shaking ata velocity of 120 rpm. Absorbance of the obtained culture broth wasmeasured (absorbance was measured by using DU640 SPECTROPHTOMETER,BECKMAN COULTER), the culture broth was inoculated into the SD medium(400 ml in a 2 L-volume conical flask with baffle fins), so that OD660was 0.01 at the start of the culture, and the yeast cells werecultivated at 30° C. for 18 hours with shaking by rotation at a velocityof 120 rpm. Then, γ-Glu-Abu was added at 10 ppm or 100 ppm as the finalconcentration for the experiment utilizing delayed addition of γ-Glu-Abuto the medium, or no substance was added for the control, and theculture was continued for further 1 hour (total culture time was 19hours). Extract was obtained from the obtained culture broth in the samemanner as that of Example 3, and contents of the compounds in the cellswere measured.

As shown in Table 7, it was found that γ-Glu-Abu in the medium could beuptaken into the cells and accumulated in the cells as in Example 4,even when γ-Glu-Abu was added afterward. Moreover, not only γ-Glu-Abu,but also γ-Glu-Abu-Gly accumulated, as in Examples 3 and 4.

TABLE 7 γ-Glu-Abu γ-Glu-Abu-Gly (ppm) (ppm) SD medium 21 39 Medium addedγ-Glu-Abu (10 ppm) 2330 1430 afterward Medium added γ-Glu-Abu (50 ppm)12545 3106 afterward Medium added γ-Glu-Abu (100 ppm) 10241 2055afterward

Example 6 γ-Glu-Abu Content Based on Solid Content of Yeast Extract

Solid contents of the extracts obtained in Examples 3 to 5 weremeasured, and γ-Glu-Abu contents based on the solid contents of theextracts were calculated from the γ-Glu-Abu contents based on dry cellweight. As a result, it was found that the γ-Glu-Abu content markedlyincreased compared with commercially available yeast extracts.

TABLE 8 γ-Glu-Abu (ppm) SD medium 123 Medium containing Abu (10 ppm)1013 Medium containing Abu (50 ppm) 5068 Medium containing Abu (100 ppm)8250 Medium containing γ-Glu-Abu (10 ppm) 8970 Medium containingγ-Glu-Abu (100 ppm) 97316 Medium added γ-Glu-Abu (10 ppm) afterward11639 Medium added γ-Glu-Abu (50 ppm) afterward 62104 Medium addedγ-Glu-Abu (100 ppm) afterward 55382

Example 7 Effect of Addition of Abu to Candida utilis Type Strains, NBRC10707 Strain and NBRC 0988 Strain

Then, effect of addition of Abu to the Candida utilis type strains, NBRC10707 strain and NBRC 0988 strain, was investigated. These strains aredeposited at the independent administrative agency, National Instituteof Technology and Evaluation, Biological Resource Center (NBRC, NITEBiological Resource Center, 2-5-8 Kazusakamatari, Kisarazu-shi,Chiba-ken, 292-0818, Japan) with the number of NBRC 10707 and NBRC 0988,and can be provided therefrom.

One loop each of the NBRC 10707 strain and NBRC 0988 strain wasinoculated into the SD medium (50 ml in a 500 ml-volume Sakaguchiflask), and cultivated at 30° C. for 24 hours with shaking at a velocityof 120 rpm. Absorbance of the obtained culture broth was measured(absorbance was measured by using DU640 SPECTROPHTOMETER, BECKMANCOULTER), the culture broth was inoculated into the SDP medium (400 mlin a 2 L-volume conical flask with baffle fins, corresponding to the SDmedium described in Example 3, provided that 5 g of ammonium sulfate wasreplaced with 1 g of proline at the time of preparing Yeast NitrogenBase of 10-fold concentration), so that OD660 was 0.01 at the start ofthe culture, and the yeast cells were cultivated at 30° C. with shakingby rotation at a velocity of 120 rpm. The NBRC 10707 strain wascultivated for 46.5 hours, and the NBRC 0988 strain was cultivated for22.5 hours to attain the logarithmic phase for each strain. Then, Abuwas added at a final concentration of 100 ppm, and the culture wascontinued for further 1 hour. As a control group, cultivation wascontinued for 1 hour without adding the compound. The absorbance of theculture broth to which Abu was added afterward was about 4.5 after theculture, and residual saccharides were also detected. An extract wasobtained from the obtained culture broth in the same manner as that ofExample 3, and contents of the compounds in the cells were measured. Theresults are shown in Tables 9 and 10. As a result, without the additionof Abu, γ-Glu-Abu was not detected in the cells, but with addition ofAbu, γ-Glu-Abu was detected in the cells. Also from these results, itwas found that supply of Abu was important for accumulation ofγ-Glu-Abu.

TABLE 9 NBRC 10707 strain Abu γ-Glu-Abu γ-Glu-Abu-Gly content(ppm)content (ppm) content (ppm) Abu not added 0 0 3 Abu added 2004 247 1094

TABLE 10 NBRC 0988 strain γ-Glu-Abu content γ-Glu-Abu-Gly (ppm) content(ppm) Abu not added 0 2 Abu added 107 601

Example 8 Analysis of Substrate Specificity of Yeast GSH1

Since it was found that γ-Glu-Abu was produced by a certain enzymaticreaction using Abu as a substrate in the yeast cells on the basis of theresults of Example 3, possibility of side reaction withγ-glutamylcysteine synthetase was investigated.

1) Construction of Plasmid pET-GSH1 for Expression of Yeast-Derivedγ-Glutamylcysteine Synthetase Gene (GSH1)

A plasmid pET-GSH1 for expression of the GSH1 gene coding forγ-glutamylcysteine synthetase of the Candida utilis ATCC 22023 strainwas constructed by the following procedures, and introduced intoEscherichia coli. This strain is deposited at the American Type CultureCollection (12301 Parklawn Drive, Rockville, Md. 20852, United States ofAmerica) with the number of ATCC 22023, and can be provided therefrom.

(1-1) Construction of Plasmid pAUR-GSH1 for Expression of Yeast GSH1

First, a plasmid pAUR-GSH1 for expression of yeast GSH1 was constructedby the following procedure. The construction was entrusted to TakaraBio.

By PCR using Primer G (SEQ ID NO: 3) and Primer H (SEQ ID NO: 4), whichwere produced on the basis of the nucleotide sequence (SEQ ID NO: 1) ofthe GSH1 gene of the Candida utilis ATCC 22023 strain, as well as thechromosomal DNA of the ATCC 22023 strain as the template, the sequencecontaining the GSH1 gene was amplified. Primer G consisted of a regioncontaining the start codon of the GSH1 gene in the chromosomal DNA ofthe ATCC 22023 strain, to which the KpnI recognition sequence and apartial sequence of the yeast expression plasmid pAUR123 (Takara Bio)were added at the 5′ end. Primer H consisted of a nucleotide sequencecomplementary to the C-terminal nucleotide sequence of the GSH1 gene, towhich a nucleotide sequence complementary to the sequence coding for theHis-tag, a nucleotide sequence complementary to the stop codon (TAA),the XbaI recognition sequence, and a partial sequence of pAUR123 wereadded. PCR was performed by using PrimeSTAR Max DNA Polymerase (TakaraBio) according to the protocol described in the attached manual. Theamplified fragment was introduced into pAUR123 (Takara Bio) at theKpnI-XbaI site by using In-Fusion Advantage PCR Cloning Kit (Takara Bio)to construct the plasmid pAUR-GSH1 for expression of yeast GSH1.

(1-2) Construction of Plasmid pET-GSH1 for Expression of GSH1 ofEscherichia coli

Then, a GSH1 expression plasmid pET-GSH1 for Escherichia coli wasconstructed by the following procedure.

Primer I (SEQ ID NO: 5) and Primer J (SEQ ID NO: 6) were purchased fromJapan Bio Service, which were prepared on the basis of the nucleotidesequence of the GSH1 gene of the Candida utilis ATCC 22023 strain (SEQID NO: 1). Primer I consisted of a region containing the start codon ofthe GSH1 gene in the chromosomal DNA of the Candida utilis ATCC 22023strain, to which a nucleotide sequence containing the SpeI recognitionsequence was added at the 5′ end. Primer J consisted of a nucleotidesequence complementary to the nucleotide sequence outside from the stopcodon of the GSH1 gene in pAUR-GSH1 mentioned above, to which anucleotide sequence containing the XhoI recognition sequence was addedat the 5′ end.

By PCR using Primer I and Primer J, as well as the aforementionedpAUR-GSH1 as the template, the sequence containing the GSH1 gene wasamplified. PCR was performed by preparing 50 μl of a reaction mixturecontaining the plasmid DNA, 0.2 μmol/L each of the primers, 1.25 unitsof PrimeSTAR HS DNA Polymerase (Takara Bio), 10 μL of the 5× PrimeSTARbuffer (Takara Bio), and 2.5 mmol/L each of dNTPs (dATP, dGTP, dCTP, anddTTP), and subjecting the reaction mixture to warming at 98° C. for 10seconds, then 30 cycles of 98° C. for 10 seconds, 56° C. for 5 secondsand 72° C. for 2 minutes, and further warming at 72° C. for 1 minute.

The reaction mixture after PCR (3 μl) was subjected to agarose gelelectrophoresis to confirm that a DNA fragment of about 2.0 kbcorresponding to the GSH1 gene fragment was amplified, and then the DNAfragment was purified from the remaining reaction mixture by usingEthachinmate (NIPPON GENE), and dissolved in 25 μl of dH₂O. Then, theDNA fragment in the whole volume of the obtained DNA solution wasdigested with the restriction enzymes SpeI and XhoI, then purified byusing MinElute Reaction Cleanup Kit (QIAGEN), and dissolved in 15 μl ofBuffer EB (10 mM Tris-HCl, pH 8.5, QIAGEN).

The expression plasmid pET-21a(+) (1 μg, Novagen) was digested with therestriction enzymes NheI and XhoI, then purified by using MinEluteReaction Cleanup Kit, and dissolved in 15 μl of Buffer EB. Then, the DNAfragment in the whole volume of the obtained DNA solution wasdephosphorylated with an alkaline phosphatase (calf intestine alkalinephosphatase, CIAP), then purified by using MinElute Reaction CleanupKit, and dissolved in 10 μl of Buffer EB.

The DNA fragment of about 2.0 kb containing the GSH1 gene obtainedabove, and the DNA fragment of about 5.4 kb of the expression plasmidpET-21a(+) (Novagen) obtained above were reacted at 16° C. for 30minutes by using TaKaRa Ligation Kit Ver. 2.1 (Takara Bio), and therebyligated. Competent cells of the Escherichia coli DH5α strain (TakaraBio) were transformed by the heat shock method using the above reactionmixture, and the transformants were applied on the LB [10 g/L of Bactotryptone (Difco), 5 g/L of yeast extract (Difco), and 5 g/L of sodiumchloride (Wako)] agar medium containing 100 μg/ml of ampicillin, andcultured overnight at 37° C.

From the grown colonies of the transformants, a plasmid was extracted bya known method, and the nucleotide sequence thereof was determined by aknown method. The obtained plasmid was a plasmid consisting of the GSH1gene derived from the Candida utilis ATCC 22023 strain having thesequence coding for the His-tag at the 3′ end, which was ligated to T7promoter on the downstream side, and this plasmid was designatedpET-GSH1. The nucleotide sequence of the GSH1 gene derived from theCandida utilis ATCC 22023 strain and the amino acid sequence encodedthereby are shown in SEQ ID NOS: 1 and 2, respectively.

Then, competent cells of the Escherichia coli Rosetta2(DE3)pLysS strain(Novagen) were transformed with pET-GSH1 by the heat shock method, andthe transformants were applied on the LB agar medium containing 100μg/ml of ampicillin and 30 μg/ml of chloramphenicol, and culturedovernight at 37° C. Plasmids were extracted from the grown colonies ofthe transformants in a known manner, and the structures thereof wereanalyzed by using restriction enzymes to confirm that the transformantsharbored pET-GSH1. The Escherichia coli Rosetta2(DE3)pLysS strainharboring pET-GSH1 was designated Escherichia coliRosetta2(DE3)pLysS/pET-GSH1.

2) Purification of C-Terminal His-Tag-Added Recombinant Gsh1

Escherichia coli Rosetta2(DE3)pLysS/pET-GSH1 obtained as described abovewas inoculated into 3 mL of the LB medium containing 100 μg/ml ofampicillin and 30 μg/ml of chloramphenicol contained in a test tube, andcultured at 37° C. for 16 hours with shaking. The obtained culture broth(2 ml) was inoculated into 100 ml of the LB medium contained in aSakaguchi flask. Culture was performed at 37° C. for 2 hours withshaking, then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at afinal concentration of 0.5 mmol/L, and culture was further continued at30° C. for 4 hours. The culture broth was centrifuged to obtain wetcells.

The wet cells were suspended in 10 ml of a 100 mmol/L Tris-hydrochloricacid buffer (pH 8.0) containing 300 mM sodium chloride, the cells weredisrupted by ultrasonication, and the suspension was centrifuged. Fromthe obtained supernatant, His-tag added recombinant Gsh1 was purified byusing a His-tag-added protein purification kit, Ni Sepharose 6 Fast Flow(GE Healthcare), according to the protocol described in the attachedmanual, and then desalted by using PD-10 column (GE Healthcare)according to the protocol described in the attached manual. Thispurified and desalted Gsh1 was used for the following experiments aspurified Gsh1.

3) Analysis of Substrate Specificity of GSH1

A reaction mixture (pH 8.0, 200 μl) containing the purified recombinantGSH1 obtained above (24.6 μg), 100 mmol/L of Tris-HCl (pH 8.0), 12.5mmol/L of Abu, 12.5 mmol/L of glutamic acid, 12.5 mmol/L of adenosinetriphosphate (ATP), 12.5 mmol/L of magnesium sulfate, and 2 mmol/L ofdithiothreitol (DTT) was prepared, and the reaction was performed at 37°C. for 16 hours.

After completion of the reaction, the reaction product was analyzed byHPLC. The analysis conditions were as follows.

(1) HPLC: HITACHI L-2000 Series

(2) Isolation column: Synergi 4μ Hydro-RP 80A; internal diameter, 4.6mm; length, 250 mm; particle size, 4 μm (Phenomenex)(3) Column temperature: 40° C.(4) Mobile phase A: 50 mM phosphate buffer (pH 2.5)(5) Mobile phase B: acetonitrile(6) Flow rate: 1.0 ml/minute(7) Elution conditions: Elution was performed by using a mixture of themobile phase A and the mobile phase B. The ratio of the mobile phase Bin the mixture was as follows: 0 minute (0%), 0 to 5 minutes (0 to2.5%), 5 to 15 minutes (2.5%), 15 to 30 minutes (2.5 to 40%), 30 to 30.1minutes (40 to 0%), and 30.1 to 50 minutes (0%).

(8) Detection: UV 210 nm

As a result of the aforementioned measurement, the retention time of thepeak of the reaction product agreed with that of a γ-Glu-Abu sample, andit was judged that the product was γ-Glu-Abu. As a result ofquantification, γ-Glu-Abu concentration was found to be 10.6 mM.

These results revealed that GSH1 of yeast recognized Abu as a substrate.

Example 9 Effect of Addition of Abu to Saccharomyces cervisiae GSH1Expression-Enhanced Strain

Since it was revealed by the investigation performed in Example 8mentioned above based on in vitro enzymatic reaction that GSH1 wasresponsible for an enzymatic reaction using Abu and Glu as substrates,it was then investigated whether this reaction would actually occur inthe yeast cells.

1) Acquisition of Uracil Auxotrophic Strain (Ura3 Mutant)

A uracil auxotrophic strain was obtained by introducing anURA3-neighboring DNA except for the URA3 gene into a Saccharomycescervisiae wild-type strain monoploid (Matα type), and disrupting theURA3 gene, as shown below.

First, a 500-bp upstream region of URA3 was amplified by PCR using theprimers of SEQ ID NO: 7 (gataaggaga atccatacaa) and SEQ ID NO: 8(gtgagtttag tatacatgca tttacttata atacagtttt gatttatctt cgtttcctgc), andthe chromosomal DNA of the aforementioned wild-type strain as thetemplate. Furthermore, a 500-bp downstream region of URA3 was alsoamplified using the primers of SEQ ID NO: 9 (aaaactgtat tataagtaaa) andSEQ ID NO: 10 (cacttatttg cgatacagaa). As for PCR conditions, a cycleconsisting of thermal denaturation at 94° C. for 10 seconds, annealingat 55° C. for 10 seconds, and extension at 72° C. for 1 minute wasrepeated 25 times. Then, overlap PCR was performed by using the abovetwo kinds of DNA fragments purified by ethanol precipitation astemplates and the primers of SEQ ID NO: 11 (gataaggaga atccatacaa) andSEQ ID NO: 12 (cacttatttg cgatacagaa) to obtain a 1-kb DNA fragmentconsisting of the 500-bp upstream region and 500-bp downstream region ofthe URA3 gene ligated together. The wild-type strain was transformedwith this DNA fragment, and then cultured overnight in the SD medium towhich uracil was added, and the cells were applied to 5-FOA platemedium. The ura3Δ0 strain was obtained from the resulting transformants.This strain was given a private number AJ14956, and was deposited at theindependent administrative agency, Agency of Industrial Science andTechnology, International Patent Organism Depository (Tukuba Central 6,1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug.18, 2010, and assigned an accession number of FERM P-22000. Then, thedeposit was converted to an international deposit under the provisionsof the Budapest Treaty, and assigned an accession number of FERMBP-11299.

2) Preparation of Template Plasmid for Promoter Substitution

First, the URA3 locus was amplified by PCR using primers of SEQ ID NO:13 (atagcatgct cataaaattg ataaggaga) and SEQ ID NO: 14 (atagaattcaggacgtcatt agtggcgaa) and the chromosomal DNA of a Saccharomycescerevisiae wild-type strain as the template (thermal denaturation: 94°C. for 10 seconds, annealing: 50° C. for 10 seconds, extension: 72° C.for 1 minute, 25 cycles). The resulting DNA fragment was purified byethanol precipitation, and then digested with SphI and EcoRI, and theproduct was inserted into the plasmid pUC19 at the SphI-EcoRI sites toobtain pUC19-URA3. Then, the ADH1 promoter region was amplified from thechromosomal DNA of the Saccharomyces cerevisiae wild-type strain usingthe primers of SEQ ID NO: 15 (atactgcaga taatcgatta attttttttt ctttc)and SEQ ID NO: 16 (atactgcaga agtagataat tacttcctt). This DNA fragmentwas digested with PstI, and inserted into pUC19-URA3 digested with PstIand treated with CIAP at the PstI site to obtain pUC19-ADH1p-URA3. Itwas confirmed that ADH1p was correctly inserted in the forward directionwith respect to the URA3 gene by sequencing the neighboring regionthereof. In a similar manner, the ADH1 promoter amplified by using theprimers of SEQ ID NO: 17 (atagacgtct aatttttttt tctttc) and SEQ ID NO:18 (atagacgtct gttttatatt tgttgtaaa) was digested with AatII, andinserted into pUC19-ADH1p-URA digested with AatII and treated with CIAPat the AatII site to obtain pUC19-ADH1p-URA3-ADH1p. It was confirmedthat ADH1p was correctly inserted in the forward direction with respectto the URA3 gene by sequencing the neighboring region thereof.

3) Introduction of ADH1 Promoter into GSH1 Gene on Chromosome

PCR was performed by using the primer of SEQ ID NO: 19(TATTGCCCCAGTGTTCCCTCAACAACCTTGGTAGTTGGAGCGCAATTAGCGTATCCTGTACCATACTAATTCTCTTCTGCTCTTAACCCAACTGCACAGA), which has a GSH1 upstreamsequence at the 5′ end, the primer of SEQ ID NO: 20(ATACCTTCATCCCTTATGTGTTCATTGTACGTCCTAGACTCAAACCACTGCAAAGGCGTGCCCAAAGCTAAGAGTCCCATTGTATATGAGATAGTTGATT), which has a part of asequence in ORF starting from the start codon of the GSH1 gene, andpUC19-ADH1p-URA3-ADH1p as the template (thermal denaturation: 94° C. for10 seconds, annealing: 60° C. for 10 seconds, extension: 72° C. for 4minutes) to prepare a DNA fragment having URA3 between ADH1p promoters.The ura3Δ0 strain was transformed with this DNA fragment, and plated onan SD plate medium to obtain transformants, and a strain in which theGSH1 promoter was replaced with the ADH1 promoter-URA3-ADH1 promoter wasobtained from the transformants.

4) Elimination of URA3 Selective Marker and Substitution of Promoter forGSH1 Gene

The strain in which the ADH1p promoter-URA3-ADH1 substitutes for theGSH1 promoter was cultured overnight in a uracil-supplemented SD medium,and an appropriate volume of the culture was applied to 5-FOA platemedium. From the grown colonies, a strain in which URA3 was removed, andthe GSH1 promoter was replaced with the ADH1 promoter by homologousrecombination between the introduced ADH1 promoters, AG1-ura3Δ0 strain,was obtained. Furthermore, by introducing a DNA amplified by using awild-type genome as the template and the primers of SEQ ID NO: 21(AGTTACAGCAATGAAAGAGCAGAGCGAGAG) and SEQ ID NO: 22(ATTACTGCTGCTGTTCCAGCCCATATCCAA) into the above strain, a strain inwhich URA3 was returned to wild-type, and the GSH1 promoter was replacedwith the ADH1 promoter, was obtained. This strain was designated AG1strain. In a similar manner, by introducing a DNA amplified by using awild-type genome as the template and the primers of SEQ ID NO: 23(AGTTACAGCAATGAAAGAGCAGAGCGAGAG) and SEQ ID NO: 24(ATTACTGCTGCTGTTCCAGCCCATATCCAA) into the ura3Δ0 strain, AJ14956, astrain in which URA3 was returned to wild-type was obtained. This strainwas designated Control strain.

5) Effect of Addition of Abu to Control Strain and AG1 Strain

Then, one loop each of the Control strain and the AG1 strain wasinoculated into the SD medium (50 ml in a 500 ml-volume Sakaguchiflask), and cultivated at 30° C. for 24 hours with shaking at a velocityof 120 rpm. Absorbance of the obtained culture broth was measured, theculture broth was inoculated into the SD medium containing Abu atvarious concentrations (50 ml in a 500 ml-volume Sakaguchi flask) sothat OD660 was 0.01 at the start of the culture, and the yeast cellswere cultivated at 30° C. for 19 hours with shaking at a velocity of 120rpm. Cells corresponding to 20 OD units were obtained from the resultingculture broth by centrifugation. Thereafter, an extract was obtained,and intracellular contents of the compounds were measured in the samemanner as that of Example 3

As a result, as shown in the following tables, in the AG1 strain, theγ-Glu-Abu content markedly increased compared with the Control strain.On the basis of this result, it was revealed that GSH1 recognized Abu asa substrate also in the cells and produced γ-Glu-Abu from Abu. Inaddition, when the AG1 strain was cultivated in the SD medium notcontaining Abu, intracellular γ-Glu-Abu content was lower than thedetection limit, and therefore it was also found that the intracellularγ-Glu-Abu content was not necessarily increased only by the enhancementof expression of GSH1, and supply of Abu was important.

TABLE 11 Effect of addition of Abu for Control strain Abu γ-Glu-Abuγ-Glu-Abu-Gly (ppm) (ppm) (ppm) SD medium 33 32 5 Medium containing Abu119 83 51 (10 ppm) Medium containing Abu 636 556 240 (50 ppm) Mediumcontaining Abu 2442 1243 488 (100 ppm)

TABLE 12 Effect of addition of Abu for AG1 strain Abu γ-Glu-Abuγ-Glu-Abu-Gly (ppm) (ppm) (ppm) SD medium 21 ND ND Medium containing Abu110  3540  684 (10 ppm) Medium containing Abu 250 16012 1233 (50 ppm)Medium containing Abu 353 23380 1427 (100 ppm)

Example 10 Effect of Addition of Abu to GSH1 Expression-Enhanced Strainof Candida utilis

Effect of addition of Abu to a GSH1 expression-enhanced strain ofCandida utilis can also be confirmed in the same manner as that ofExample 7. Specifically, by using Candida utilis NBRC 0988 as a parentstrain and the known Cre-loxP system, an uracil auxotrophic CUD4F strainin which the URA3 gene on the chromosome is deleted can be obtained(Shigeru Ikushima et al., 2009, Biosci. Biotechnolo. Biochem., 73(4),879-884). Since information on the gene sequences required for thegenetic manipulation is described in WO95/32289, the paper of U.Gueldener et al. (Nucleic Acids Research, 2002, Vol. 30, No. 6, e23),the paper of Gritz L. and Davis J. (Gene, 25, 179-188 (1983)), and soforth, various tools may be prepared on the basis of such sequenceinformation.

A plasmid for expression of GSH1 of Candida utilis can be constructed bya known method as follows. The known plasmid pRI177 (Ryo Iwakiri et al.,2005, Yeast, 22, 1079-1087) can be digested with the restriction enzymeBamHI and purified in a conventional manner to obtain a linear plasmid.In addition, the method for preparing the plasmid YRpGAP, which isequivalent to the plasmid pR177, is also disclosed in Japanese PatentLaid-open (Kokai) No. 2006-75122.

Further, the ORF region of GSH1 is amplified by PCR using the plasmidpAUR-GSH1 containing the sequence of GSH1 of Candida utilis, which wasconstructed in Example 9, as the template, as well as Primer S (SEQ IDNO: 25, GCAGCCCGGGGGATCATGG-GGCTGCTATCATTAGG, 15 nucleotides of the 5′end is a sequence homologous to the terminal sequence of the linearplasmid obtained by digestion with the restriction enzyme BamHI) andPrimer T (SEQ ID NO: 26, TAGAACTAGTGGATCTTAA-GCCCTTTGGGTTGTTTATC, 15nucleotides of the 5′ end is a sequence homologous to the terminalsequence of the linear plasmid obtained by digestion with therestriction enzyme BamHI). For this PCR, sequences homologous to theterminal sequences produced by digestion of pR177 with the restrictionenzyme BamHI are added to the terminuses of Primers S and T. The PCRproduct can be purified in a conventional manner, and the purified PCRproduct and the linear plasmid can be ligated by using In-FusionAdvantage PCR Cloning Kit (Takara Bio). By choosing a plasmid having theobjective sequence, autonomously replicable plasmid pCGSH1 containingthe GSH1 region of Candida utilis can be constructed. The constructionprocedure of pCGSH1 is shown in FIG. 3.

Further, the URA3 gene as the selection marker can also be introducedinto pCGSH1 mentioned above by using In-Fusion Advantage PCR CloningKit. Specifically, the full length of pCGSH1 is amplified by PCR usingPrimer U (SEQ ID NO: 27, TTACGCCAAGCGCGCAATTA) and Primer V (SEQ ID NO:28, TCATGGTCATAGCTGTTTCC) according to the protocol described in theattached manual. For this PCR, predetermined regions of the primers canbe designed according to the protocol described in the attached manual,and a linear plasmid blunt-ended at a desired position can be preparedby using them. The URA3 gene to be introduced can be amplified by PCRusing Primer W (SEQ ID NO: 29, GCGCGCTTGGCGTAACAAATAGCTCTCTACTTGCT, 15nucleotides of the 5′ end constitute a sequence homologous to theterminal sequence of the linear plasmid), and Primer X (SEQ ID NO: 30,CAGCTATGACCATGAGCAATCTACAACTTCGAAA, 15 nucleotides of the 5′ endconstitute a sequence homologous to the terminal sequence of the linearplasmid), which can be designed on the basis of the known sequenceinformation (Luis Rodriguez et al., 1998, Yeast, 14, 1399-1406), as wellas the genome of the NBRC 0988 strain as the template. In this PCR, byadding terminal sequence of the linear plasmid to the terminuses of theprimers, the gene can be ligated with the plasmid by using In-FusionAdvantage PCR Cloning Kit. As described above, an autonomouslyreplicable vector pCGSH1-URA3 for expression of GSH1 containing the URA3gene as the selection marker can be constructed. Outline of theconstruction procedures of pCGSH1-URA3 is shown in FIG. 4.

Then, pCGSH1-URA3 is introduced into the CUD4F strain by the knownelectroporation method for Candida utilis (Shigeru Ikushima et al.,2009, Biosci. Biotechnolo. Biochem., 73(4), 879-884). By spreading theobtained transformants on the SD medium and selecting a transformanthaving the objective plasmid from the grown strains, a GSH1expression-enhanced strain can be obtained. If this GSH1expression-enhanced strain is cultivated in the SDP medium containingAbu in the same manner as that of Example 7, γ-Glu-Abu is accumulated inthe cells.

Example 11 Analysis of Substrate Specificity of Yeast GSH2

Since it was found that a part of accumulated γ-Glu-Abu is metabolizedinto γ-Glu-Abu-Gly by a certain enzymatic reaction using γ-Glu-Abu as asubstrate in yeast cells on the basis of the results of Examples 3, 4,and 5, possibility of side reaction of glutathione synthetase wasinvestigated.

1) Construction of Plasmid pET-GSH2 for Expression of GlutathioneSynthetase Gene (GSH2) Derived from Yeast

An expression plasmid pET-GSH2 for the GSH2 gene coding for glutathionesynthetase of the Saccharomyces cervisiae S288C strain was constructedby the following procedures, and introduced into Escherichia coli.

(1-1) Construction of Plasmid pAUR-GSH2 for Expression of Yeast GSH2

First, an expression plasmid pAUR-GSH2 for yeast was constructed by thefollowing procedure. The construction was entrusted to Takara Bio.

By PCR using Primer A (SEQ ID NO: 33) and Primer B (SEQ ID NO: 34),which were produced on the basis of the nucleotide sequence (SEQ ID NO:31) of the GSH2 gene of the Saccharomyces cervisiae S288C strain, aswell as the chromosomal DNA of the S288C strain as the template, thesequence containing the GSH2 gene was amplified. Primer A consisted of aregion containing the start codon of the GSH2 gene in the chromosomalDNA of the S288C strain, to which the KpnI recognition sequence and apartial sequence of the yeast expression plasmid pAUR123 (Takara Bio)were added at the 5′ end. Primer B consisted of a nucleotide sequencecomplementary to the C-terminal nucleotide sequence of the GSH2 gene, towhich a nucleotide sequence complementary to the sequence coding for theHis-tag, a nucleotide sequence complementary to the stop codon (TAA),the XbaI recognition sequence, and a partial sequence of pAUR123 wereadded. PCR was performed by using PrimeSTAR Max DNA Polymerase (TakaraBio) according to the protocol described in the attached manual. Theamplified fragment was introduced into the expression plasmid pAUR123for yeast (Takara Bio) at the KpnI-XbaI site by using In-FusionAdvantage PCR Cloning Kit (Takara Bio) to construct the expressionplasmid pAUR-GSH2 for expression of GSH2 in yeast.

(1-2) Construction of Plasmid pET-GSH2 for Expression of GSH2 inEscherichia coli

Then, a GSH2 expression plasmid pET-GSH2 for Escherichia coli wasconstructed by the following procedure.

Primer C (SEQ ID NO: 35) and Primer D (SEQ ID NO: 36) were purchasedfrom Japan Bio Service, which were produced on the basis of on thenucleotide sequence of the GSH2 gene of the Saccharomyces cervisiaeS288C strain. Primer C consisted of a region containing the start codonof the GSH2 gene in the chromosomal DNA of the Saccharomyces cervisiaeS288C strain, to which a nucleotide sequence containing the NdeIrecognition sequence was added at the 5′ end. Primer D consisted of anucleotide sequence complementary to the nucleotide sequence outsidefrom the stop codon of the GSH2 gene in pAUR-GSH2 mentioned above, towhich the XhoI recognition sequence was added at the 5′ end.

By PCR using Primer C and Primer D, as well as the aforementionedpAUR-GSH2 as the template, the sequence containing the GSH2 gene wasamplified. PCR was performed by preparing 50 μl of a reaction mixturecontaining the plasmid DNA, 0.2 μmol/L each of the primers, 1.25 unitsof PrimeSTAR HS DNA Polymerase (Takara Bio), 10 μL of the 5× PrimeSTARbuffer (Takara Bio), and 2.5 mmol/L each of dNTPs (dATP, dGTP, dCTP, anddTTP), and subjecting the reaction mixture to warming at 98° C. for 10seconds, then 30 cycles of 98° C. for 10 seconds, 56° C. for 5 secondsand 72° C. for 2 minutes, and further warming at 72° C. for 1 minute.

The reaction mixture after PCR (3 μl) was subjected to agarose gelelectrophoresis to confirm that a DNA fragment of about 1.5 kbcorresponding to the GSH2 gene fragment was amplified, and then the DNAfragment was purified from the remaining reaction mixture by usingEthachinmate (NIPPON GENE), and dissolved in 25 μl of dH₂O. Then, theDNA fragment in the whole volume of the obtained DNA solution wasdigested with the restriction enzymes NdeI and XhoI, then purified byusing MinElute Reaction Cleanup Kit (QIAGEN), and dissolved in 15 μl ofBuffer EB (10 mM Tris-HCl, pH 8.5, QIAGEN).

The expression plasmid pET-21a(+) (1 μg, Novagen) was digested with therestriction enzymes NdeI and XhoI, then purified by using MinEluteReaction Cleanup Kit, and dissolved in 15 μl of Buffer EB. Then, the DNAfragment in the whole volume of the obtained DNA solution wasdephosphorylated with an alkaline phosphatase (calf intestine alkalinephosphatase, CIAP), purified by using MinElute Reaction Cleanup Kit, anddissolved in 10 μl of Buffer EB.

The DNA fragment of about 1.5 kb containing the GSH2 gene obtainedabove, and the DNA fragment of about 5.4 kb of the expression plasmidpET-21a(+) obtained above were reacted at 16° C. for 30 minutes by usingTaKaRa Ligation Kit Ver. 2.1 (Takara Bio), and thereby ligated.Competent cells of the Escherichia coli DH5α strain (Takara Bio) weretransformed by the heat shock method using the above reaction mixture,and the transformants were applied on the LB [10 g/L of Bacto tryptone(Difco), 5 g/L of yeast extract (Difco), and 5 g/L of sodium chloride(Wako)] agar medium containing 50 μg/ml of ampicillin, and culturedovernight at 37° C.

From the grown colonies of the transformants, a plasmid was extracted bya known method, and the nucleotide sequence thereof was determined by aknown method. The obtained plasmid was a plasmid consisting of the GSH2gene derived from the Saccharomyces cervisiae S288C strain having thesequence coding for the His-tag at the 3′ end, which was ligated to theT7 promoter on the downstream side, and this plasmid was designatedpET-GSH2. The nucleotide sequence of the GSH2 gene derived from theSaccharomyces cervisiae S288C strain and the amino acid sequence encodedthereby are shown in SEQ ID NOS: 31 and 32, respectively.

Then, competent cells of the Escherichia coli BL21(DE3) strain (Novagen)were transformed with pET-GSH2 by the heat shock method, and thetransformants were applied on the LB agar medium containing 50 μg/ml ofampicillin, and cultured overnight at 37° C. Plasmids were extractedfrom grown colonies of the transformants in a known manner, and thestructures thereof were analyzed by using restriction enzymes to confirmthat the transformants harbored pET-GSH2. The Escherichia coli BL21(DE3)strain harboring pET-GSH2 was designated Escherichia coliBL21(DE3)/pET-GSH2.

2) Purification of C-Terminus His-Tag-Added Recombinant Gsh2

Escherichia coli BL21(DE3)/pET-GSH2 obtained as described above wasinoculated into 3 mL of the LB medium containing 100 μg/ml of ampicillinin a test tube, and cultured at 37° C. for 16 hours with shaking. Theobtained culture broth (2 ml) was inoculated into 100 ml of the LBmedium contained in a test tube. Culture was performed at 37° C. for 2hours with shaking, then isopropyl-β-D-thiogalactopyranoside (IPTG) wasadded at a final concentration of 0.5 mmol/L, and culture was furthercontinued at 30° C. for 4 hours. The culture broth was centrifuged toobtain wet cells.

The wet cells were suspended in 10 ml of a 100 mmol/L Tris-hydrochloricacid buffer (pH 8.0) containing 300 mM sodium chloride, the cells weredisrupted by ultrasonication, and the suspension was centrifuged. Fromthe obtained supernatant, His-tag-added recombinant Gsh2 was purified byusing a His-tag-added protein purification kit, Ni Sepharose 6 Fast Flow(GE Healthcare), according to the protocol described in the attachedmanual, and then desalted by using PD-10 column (GE Healthcare)according to the protocol described in the attached manual. Then, thissample was concentrated by using a centrifugal filtration membrane of 10kDa cutoff (Amicon Ultra-0.5 mL 10K (catalog number, UFC501096),MILLIPORE) according to the protocol described in the attached manual.This purified, desalted and concentrated Gsh2 was used for the followingexperiments as purified Gsh2.

3) Production of γ-Glu-Abu-Gly Using Purified Gsh2

By using the purified Gsh2 obtained above, possibility of production ofγ-Glu-Abu-Gly using γ-Glu-Abu as the substrate was examined. A reactionmixture having the following composition was prepared, and the enzymaticreaction was performed at 30° C. for 22 hours.

[Composition of Reaction Mixture]

Purified Gsh2 300 μg/500 μl Tris-HCl (pH 8.0) 100 mmol/L  γ-Glu-Abu 10mmol/L Glycine 10 mmol/L Adenosine triphosphate (ATP) 10 mmol/L MgCl₂ 10mmol/L Dithiothreitol (DTT) 0.1 mmol/L 

After completion of the reaction, the reaction product was analyzed byHPLC under the same conditions as those used in Example 8. As a result,the retention time of the peak of the reaction product agreed with thatof a γ-Glu-Abu-Gly sample, and it was judged that the product wasγ-Glu-Abu-Gly. As a result of quantification, γ-Glu-Abu-Glyconcentration was found to be about 10 mM.

Example 12 Effect of Addition of Abu to GSH2-Disrupted Strain

The results of Example 11 revealed that γ-Glu-Abu served a substrate ofglutathione synthetase of yeast to generate γ-Glu-Abu-Gly. On the otherhand, the results of Examples 4 and 5 revealed that a part ofintracellularly incorporated γ-Glu-Abu was converted into γ-Glu-Abu-Gly,but γ-Glu-Abu was also accumulated. On the basis of these results, itwas considered that all of γ-Glu-Abu accumulated in the cells could notbe metabolized into γ-Glu-Abu-Gly with the GSH2 activity of the yeastwild strain, but a lower activity thereof might provide largeraccumulation of γ-Glu-Abu. Therefore, effect of addition of Abu to aGSH2-disrupted strain was investigated.

Specifically, Saccharomyces cervisiae S288C gsh2Δ0 strain was obtainedby the following procedure. First, a region containing GSH2 includingreplacement with the kanamycin resistance gene cassette KanMX wasamplified by using the primers of SEQ ID NO: 37 (CTAGTGAAAAACAAGAAGTA)and SEQ ID NO: 38 (GCCACATAGAAAAATCGATG) as well as the genome ofGSH2-disrupted strain of YEAST KNOCK OUT STRAIN COLLECTION (Funakoshi,YCS1056) as the template. As for PCR conditions, a cycle consisting ofthermal denaturation at 94° C. for 10 seconds, annealing at 55° C. for10 seconds, and extension at 72° C. for 3 minutes was repeated 25 times.Then, the DNA fragment was purified by ethanol precipitation, and thenused to transform the S288C strain, and the cells were applied to a YPDplate medium containing G418. From the obtained transformants, thegsh2Δ0 strain was obtained.

Then, effect of addition of Abu to this strain was investigated in thesame manner as that used in Example 3. First, one loop of the gsh2Δ0strain was inoculated into the SD medium (50 ml in 500 ml-volumeSakaguchi flask), and cultured at 30° C. for 48 hours with shaking at avelocity of 120 rpm. Absorbance of the obtained culture broth wasmeasured, the culture broth was inoculated into the SD medium or the SDmedium containing 100 ppm of Abu as a final concentration (400 ml in a 2L-volume conical flask with baffle fins), so that OD660 was 0.01 at thestart of the culture (absorbance was measured by using DU640SPECTROPHTOMETER, BECKMAN COULTER), and culture was performed at 30° C.for 65.75 hours with shaking by rotation at a velocity of 120 rpm. Fromthe obtained culture broth, an extract was obtained, and intracellularcontent of γ-Glu-Abu was measured in the same manner as that of Example3.

As a result, when the strain was cultured in the SD medium, theintracellular γ-Glu-Abu content was 128 ppm, but when it was cultured inthe medium containing 100 ppm of Abu, the intracellular γ-Glu-Abucontent was 10333 ppm. Thus, this result also revealed that γ-Glu-Abuwas not accumulated only by deficiency of the GSH2 gene, but supply ofAbu was important.

Example 13 Effect of Addition of Abu to GSH2-Disrupted Strain (2)

By using a procedure using the cre-loxp system similar to that used fordisruption of URA3 of Candida utilis in Example 10 with changing a partof the primer sequences, a GSH2-disrupted Candida utilis CUDF4 straincan be obtained.

Specifically, by using Primer N-59 (SEQ ID NO: 39, AAGTAGCCAATACAACCAGC,sequence of from the −57th to −38th region upstream of ORF of GSH2 ofCandida utilis), Primer N60 (SEQ ID NO: 40,CTGCAGCGTACGAAGCTTCAGCTGGCGGGCCACTCACC-CACTCAACATCAC, 31 nucleotide fromthe 5′ end constitute a direct repeat for overlap PCR), Primer N-295(SEQ ID NO: 41, GCTGTTTTAGACTCGTTTGC, a region of the 244th to 263rdpositions of ORF of GSH2 of Candida utilis), Primer N-296 (SEQ ID NO:42, CTGCAGCGTACGAAGCTTCAGCTGGCGGCCAGAAGATTCAGACACCGGGA, 29 nucleotidesfrom the 5′ end constitute a direct repeat for overlap PCR), Primer N-61(SEQ ID NO: 43, ATTAGGTGATATCAGATCCACTAGTGGCCTGGTTTCTTAAGATCTATTCC, 30nucleotides from the 5′ end constitute a direct repeat for overlap PCR),and Primer N-62 (SEQ ID NO: 44, TAAATGCGGCTCCATCTATTG, region up to thenucleotide of the +18th position downstream of ORF of GSH2 of Candidautilis) instead of the primers IM-59, IM60, IM295, IM296, IM61, andIM-62 used for obtaining the CUDF4 strain described in ShigehitoIKUSHIMA et al., Biosci. Biotechnol. Biochem., 73 (4), 879-884, 2009,respectively, a cassette for disruption of GSH2 of Candida utilis can beprepared. By using this cassette for disruption in the same manner asthat described in the reference, GSH2 of the CUDF4 strain is disrupted.In addition, if 1 mM GSH and a required amount of uracil are added tothe medium in the transformation step and the culture step, theacquisition rate of the strain may be improved. Whether a strainobtained as described above is the objective GSH2-disrupted strain canbe confirmed by PCR. Further, by transforming such a GSH2-disruptedstrain with pCGSH1-URA3 in the same manner as that of Example 10, astrain of which expression of GSH1 is enhanced, and GSH2 is disruptedcan be obtained. If this strain is cultured in a medium containing Abu,a marked amount of γ-Glu-Abu is accumulated in the cells.

Example 14 Search of Intracellular Abu Synthesis Enzyme

Although the metabolic pathways of Saccharomyces cerevisiae have beenwell studied, any enzyme that biosynthesizes Abu is not known. However,the inventors of the present invention estimated that an enzyme reportedas aminotransferase for another substrate might have the activity forconverting AKB (α-ketobutyrate (α-ketobutyric acid)) into Abu, in viewof the fact that substrate recognition of aminotransferases wascomparatively ambiguous, which had been confirmed in researches usingother microorganisms. Therefore, there were bred a strain highlyexpressing BAT1, reported to be responsible for the transaminationreaction of BCAA (branched chain amino acid), and a strain highlyexpressing UGA1, reported to be responsible for the transaminationreaction of GABA (γ-aminobutyric acid).

1) Construction of BAT1 and UGA1 Expression Vectors

First, in a conventional manner, a constitutive expression promoter ofyeast, ADH1p, was introduced into the plasmid pYES2 (Invitrogen), whichis a yeast-Escherichia coli shuttle vector. Specifically, by PCR usingthe genome prepared from a yeast wild strain as the template, as well asprimers of SEQ ID NO: 45 (ATAACCGGTGGGTGTACAATATGGACTTC) and SEQ ID NO:46 (ATAAAGCTTTGTATATGAGATAGTTGATT), the promoter region of ADH1 wasamplified (a cycle consisting of thermal denaturation at 94° C. for 10seconds, annealing at 50° C. for 10 seconds, and extension at 72° C. for1 minute was repeated 25 times). The obtained DNA fragment was purifiedby ethanol precipitation, then digested with the restriction enzymesHindIII and AgeI, and inserted into the plasmid pYES2 at theHindIII-AgeI site to obtain pYES2-ADH1p.

Then, in order to insert ORF regions of the genes into this pYES2-ADH1p,amplification products of the genes were each subcloned into the pT7vector. Specifically, by PCR using the genome prepared from aSaccharomyces cervisiae wild strain as the template, as well as theprimers of SEQ ID NO: 47 (GGATCCATGTTGCAGAGACATTCC) and SEQ ID NO: 48(TCTAGATTAGTTCAAGTCGGC), or the primers of SEQ ID NO: 49(AAGCTTACAGACAAGAAACCGTC) and SEQ ID NO: 50 (TCTAGAGGCCTCGCTAATATAC),the ORF regions of BAT1 and UGA1 were amplified, respectively. Theobtained BAT1 amplification product was digested with the restrictionenzymes BamHI and XbaI, and inserted into the pT7 vector at theBamHI-XbaI site to obtain pT7-BAT1. The UGA1 amplification product wasdigested with the restriction enzymes HindIII and XbaI, and insertedinto the pT7 vector at the HindIII-XbaI site to obtain pT7-UGA1.

pT7-BAT1 obtained as described above was treated with the restrictionenzymes BamHI and XbaI, and the DNA fragment of BAT1 was purified byseparation based on electrophoresis and excision of the objective gene,and introduced into the plasmid pYES2-ADH1p at the BamHI-XbaI site.Further, pT7-UGA1 was treated with the restriction enzymes HindIII andXbaI, and the DNA fragment of UGA1 was purified by separation based onelectrophoresis and excision of the objective gene, and introduced intothe plasmid pYES2-ADH1p at the HindIII-XbaI site. As described above, aBAT1 high expression vector, pYES2-ADH1p-BAT1, and a UGA1 highexpression vector, pYES2-ADH1p-UGA1, were prepared.

The nucleotide sequences of BAT1 and UGA1 are shown in SEQ ID NOS: 51and 53, respectively. Further, the amino acid sequences encoded by thesegenes are shown in SEQ ID NOS: 52 and 54, respectively.

2) Breeding of S288Cura3Δ0 strain

In the same manner as that of Example 9, there was bred S288Cura3Δ0strain corresponding to the S288C strain in which the ORF region of theURA3 gene was deleted.

3) Breeding of Various Expression Strains

By transforming S288Cura3Δ0 bred in 2) with each of the expressionvectors constructed in 1), strains highly expressing each of the geneswere bred. Specifically, competent cells of S288Cura3Δ0 were prepared byusing Frozen EZ Yeast Transformation II Kit of Zymo Research, and eachof the expression vectors was introduced into the cells to obtainS288C/pYES2-ADH1p strain, S288C/pYES2-ADH1p-BAT1 strain, andS288C/pYES2-ADH1p-UGA1 strain.

4) Evaluation of Obtained Strains

The aforementioned strains were evaluated by culture in the SD medium inthe same manner as that of Example 3. The results are shown in Table 13.

TABLE 13 Abu γ-Glu-Abu γ-Glu-Abu-Gly content content content Control 23ppm 114 ppm 181 ppm BAT1 high 37 ppm 197 ppm 602 ppm expression strainUGA1 high 41 ppm 116 ppm 310 ppm expression strain

As a result, increase of the intracellular Abu content was observed inthe BAT1 high expression strain and the UGA1 high expression strain.Further, since Abu synthesized within the cells is used as a substratefor γ-Glu-Abu and γ-Glu-Abu-Gly, the total amounts of these three kindsof compounds were compared in terms of molar concentration. The totalamounts were calculated to be 2.46 μmol/g-DCW for the control strain,7.06 μmol/g-DCW for the BAT1 high expression strain, and 3.91 μmol/g-DCWfor the UGA1 high expression strain, and thus it was revealed that thehigh expression of the various aminotransferases provided increase ofamount of intracellular Abu-containing compounds, i.e., increase of Abubiosynthesis ability. Further, in the same manner as that used forobtaining the S288Cgsh2Δ0 strain from the S288Cura3Δ0 strain as a parentstrain in Example 12, S288Cura3Δ0gsh2Δ0 strain was obtained. Bypreparing competent cells of this strain and introducing each of theexpression vectors into the cells, S288Cgsh2Δ0/pYES2-ADH1p strain andS288Cgsh2Δ0/pYES2-ADH1p-BAT1 strain were obtained. If the latter strainis cultured in the SD medium, it accumulates γ-Glu-Abu.

Example 15 Effect of High Expression of GSH1 and High Expression ofAminotransferase

Since the effect of increasing the intracellular Abu-containingcompounds was provided by increase of the activity of aminotransferase,especially increase of the activity of BAT1, in the aforementionedinvestigation, effect of combination thereof with high expression ofGSH1 was examined. The uracil auxotrophic strain obtained in Example 9,AG1-ura3Δ0, was transformed with pYES2-ADH1p-BAT1 prepared in Example 14to breed a BAT1 and GSH1 high expression strain, AG1/pYES2-ADH1p-BAT1strain. In the same manner as that of Example 3, this strain wascultured in the SD medium, and the γ-Glu-Abu content in the cells andthe γ-Glu-Abu content in solid content of extract were calculated. As aresult, the AG1/pYES2-ADH1p-BAT1 strain contained 1813 ppm of γ-Glu-Abubased on the dry cell weight, and the extract obtained from the cells ofthe strain contained about 4560 ppm of γ-Glu-Abu based on solid content.

Example 16 Yeast Extract Added with Abu and Treated withγ-glutamyltransferase

A 1% aqueous solution of yeast extract containing regent GSH (Wako PureChemical Industries) having a GSH content of about 8% based on the solidcontent was prepared, and adjusted to pH 7.0 with NaCH. Powder Abu wasadded to this solution at a final concentration of 800 ppm, 1600 ppm, or8000 ppm in the aqueous solution to prepare test samples. Further, theyeast extract aqueous solution not containing Abu was used as a control.γ-GTP (γ-glutamyltranspeptidase from equine kidney; Sigma; code,G9270-100UN) was added to these test samples at 0.05 mg/ml, and theenzymatic reaction was allowed at 37° C. for 120 minutes. The reactionmixture was immediately cooled on ice after the reaction, and theγ-Glu-Abu content was measured. Further, the solid content was measuredby using a part of the reaction mixture, and the content of γ-Glu-Abuproduced by the enzymatic reaction based on the solid content wascalculated.

As a result, as shown in FIG. 5, γ-Glu-Abu was hardly produced in the noAbu addition experiment, but more γ-Glu-Abu was produced as the additionamount of Abu was increased.

Example 17 Organoleptic Evaluation of Yeast Extract Containing γ-Glu-Abu(1)

First, samples for organoleptic evaluation were prepared by thefollowing procedure. In the same manner as that used in Example 4, oneloop of the S288C strain was inoculated into the SD medium (50 ml in 500ml-volume Sakaguchi flask), and cultivated at 30° C. for 24 hours withshaking at a velocity of 120 rpm. Absorbance of the obtained culturebroth was measured, the culture broth was inoculated into the SD medium(400 ml in 2 L-volume conical flask with baffle fins, 4 flasks) or theSD medium containing 200 ppm of γ-Glu-Abu as a final concentration (400ml in 2 L-volume conical flask with baffle fins, 4 flasks), so thatOD660 was 0.01 at the start of the culture (absorbance was measured byusing DU640 SPECTROPHTOMETER, BECKMAN COULTER), and the yeast cells werecultivated at 30° C. for 19 hours with shaking by rotation at a velocityof 120 rpm. In the same manner as that used in Example 4, an extract wasobtained from the obtained cells, and the γ-Glu-Abu concentration in theextract, and the solid content of the extract were obtained. As aresult, the γ-Glu-Abu concentration in the extract prepared in theγ-Glu-Abu addition experiment was about 1,000 ppm, and the solid contentthereof was about 0.59% (Extract 1). On the other hand, the solidcontent of the extract prepared in the no addition experiment was about1.00% (Extract 2).

Then, kokumi of the following samples was evaluated by six specialpanelists according to the following method.

Control sample: Aqueous solution containing 0.2% of MSG, and 0.5% ofNaClSample 1: Control sample to which Extract 1 is added so that γ-Glu-Abucontent is about 40 ppmSample 2: Control sample to which Extract 2 is added so that solidcontent is the same as that of Extract 1 added to Sample 1

As a result, all the panelists evaluated that stronger kokumi of theinitial taste was obtained with Sample 1. On the basis of this result,it was confirmed that γ-Glu-Abu exhibited the characteristic thereof,initial taste, even in yeast extract.

Example 18 Organoleptic Evaluation of Yeast Extract Containing γ-Glu-Abu(2)

One loop of the Saccharomyces cervisiae AJ14892 strain (Japanese PatentLaid-open (Kokai) No. 2008-61525) that accumulates γ-glutamylcysteine(γ-GC) was inoculated into the SD medium (50 ml in 500 ml-volumeSakaguchi flask, 4 flasks), and cultivated at 30° C. for 48 hours withshaking at a velocity of 120 rpm. Absorbance of the obtained culturebroth was measured, the culture broth was inoculated into the SD medium(400 ml in a 2 L-volume conical flask with baffle fins, 4 flasks), sothat OD660 was 0.01 at the start of the culture, and the yeast cellswere cultivated at 30° C. with shaking by rotation at a velocity of 120rpm. As for the culture time, residual sugar and absorbance wereperiodically measured, and the culture was performed for about 42 hoursso that the absorbance became about 1.8, which is the absorbanceobtained when the S288C strain was cultured in the SD medium for 19hours. In the same manner as that used in Example 4, an extract wasobtained from the cells, and the solid content of the extract wereobtained. As a result, the solid content was found to be about 0.71%(Extract 3). The γ-GC content in Extract 3 was about 390 ppm. Asdescribed above, Extract 3 having a γ-GC content of about 5.5% based onthe solid content was obtained.

Then, kokumi of the following samples was evaluated by six specialpanelists according to the following method.

Control sample: aqueous solution containing 0.2% of MSG, and 0.5% ofNaClSample 3: Control sample to which Extract 3 is added so that solidcontent is the same as that of Extract 1 added to Sample 1Sample 4: Control sample to which marketed GSH-rich yeast extract(Aromild UG8, Kohjin Co., Ltd.) was added so that solid content is thesame as that of Extract 1 added to Sample 1

For the evaluation, kokumi titer of the control sample was defined as0.0, and kokumi titer of Sample 4 was defined as 3.0. As a result, asshown in the following table, it was found that the γ-Glu-Abu-containingyeast extract (Sample 1) showed an organoleptic profile different fromthose obtained with the sample similarly containing a dipeptide, theγ-GC-rich yeast extract (Sample 3), and the sample containing atripeptide, the GSH-rich yeast extract (Sample 4), and gave a highkokumi titer for the initial taste.

TABLE 14 Kokumi of Kokumi of initial taste middle-aftertaste Control 0.00.0 Sample 1 4.0 1.8 Sample 3 1.8 2.2 Sample 4 3.0 3.0

Example 19 Effect of Enhancement of α-Ketobutyric Acid-Producing Ability

Then, effect of enhancement of α-ketobutyric acid-producing ability wasexamined. Although it was not known so far that α-ketobutyric acid mightserve as a precursor of Abu within yeast cells, whether enhancement ofα-ketobutyric acid-producing ability could increase the Abu-producingability in yeast cells was examined, since activation ofaminotransferase increased the Abu-producing ability in yeast cells.

First, in order to insert the ORF region of CHA1 coding for serine(threonine) deaminase into pYES2-ADH1p prepared in Example 13, anamplification product of CHA1 was subcloned into the pT7 vector.Specifically, by PCR using the genome prepared from a Saccharomycescervisiae wild strain as the template, and the primers of SEQ ID NO: 55(ATAAAGCTTAACCAGCGAGATGTCG) and SEQ ID NO: 56(CTCTCTAGAGGGCAAATTGATGCTTC), the ORF region of CHA1 was amplified. Theobtained CHA1 amplification product was digested with the restrictionenzymes HindIII and XbaI, and inserted into the pT7 vector at theHindIII-XbaI site to obtain pT7-CHA1.

pT7-CHA1 obtained as described above was treated with the restrictionenzymes HindIII and XbaI, and the DNA fragment of CHA1 was purified byseparation based on electrophoresis and excision of the objective gene,and introduced into the plasmid pYES2-ADH1p at the HindIII-XbaI site. ACHA1 high expression vector pYES2-ADH1p-CHA1 was prepared as describedabove.

The nucleotide sequence of CHA1 is shown in SEQ ID NO: 57. Further, theamino acid sequence encoded by this gene is shown in SEQ ID NO: 58.

Then, the promoter region of BAT1 in the uracil auxotrophic strain bredin Example 9, AG1-ura3Δ0, was replaced with the promoter region of PGK1according to the method of Sofyanovich et al. (Olga A. Sofyanovich etal., A New Method for Repeated “Self-Cloning” Promoter Replacement inSaccharomyces cerevisiae, Mol. Biotechnol., 48, 218-227 (2011)). The DNAfragment for the replacement of the promoter was prepared byamplification based on PCR using the pPUP plasmid described in thereference and the primers for replacement of BAT1, SEQ ID NO: 59(GCCAGGCGGTTGATACTTTGTGCAGATTTCATACCGGCTGTCGCTATTATTACTGATGAATTGGCTCTCTTTTTGTTTAATCTTAACCCAACTGCACAGA) and SEQ ID NO: 60(TTGGATGCATCTAATGGGGCACCAGTAGCGAGTGTTCTGATGGAGAATTTCCCCAACTTCAAGGAATGTCTCTGCAACATTGTTTTATATTTGTTGTAAA). The GSH1- andBAT1-enhanced strain of the uracil auxotrophic strain constructed asdescribed above, AGB-ura3Δ0 strain, was transformed with the CHA1 highexpression vector to breed a strain highly expressing GSH1, BAT1 andCHA1. Specifically, in the same manner as that used in Example 14,competent cells of the AGB-ura3Δ0 strain were prepared by using FrozenEZ Yeast Transformation II Kit of Zymo Research, and pYES2-ADH1p-CHA1was introduced into the cells to obtain AGB-ura3Δ0/pYES2-ADH1p-CHA1strain.

The aforementioned strain was evaluated by culture in the SD medium inthe same manner as that of Example 14. As a result, this straincontained 2024 ppm of γ-Glu-Abu based on the dry cell weight.

Example 20 Effect of Disruption of Peptidase

Then, effect of disruption of an enzyme gene DUG2, reported to beinvolved in the decomposition of GSH, was investigated. The nucleotidesequence of DUG2 is shown in SEQ ID NO: 61, and the amino acid sequenceencoded by this gene is shown in SEQ ID NO: 62.

First, by using the primer of SEQ ID NO: 63 having 80 nucleotidesupstream from the start codon of DUG2(TTAAGTGAAAAACTATTTCGAGAAACCGAACAACCCTGTAAGGAAAAGTGAAAAACGAGGGCAGAAGTAATTGTGAAATCGTTCATCATCTCATGGATCT), and the primer of SEQ IDNO: 64 having 80 nucleotides downstream from the stop codon of DUG2(ACTAATTATCATTAGGTAGAGGCCTACATATGCAAATTGGGTATATATTAAGCACTTTAAAATCAATTGTTTGTAGTTGTAGATTCCCGGGTAATAACTG), the URA3 gene of a wildtype strain was amplified. As for PCR conditions, a cycle consisting ofthermal denaturation at 94° C. for 10 seconds, annealing at 50° C. for10 seconds, and extension at 72° C. for 2 minutes was repeated 25 times.The AG1-ura3Δ0 strain was transformed with the obtained DNA fragment,and applied to the SD medium not containing uracil. A dug2Δ strain ofthe AG1-ura3Δ0 (henceforth referred to as AG1-dug2Δ0 strain) wasobtained from the grown transformants. The AG1 strain and the AG1-dug2Δ0strain were cultured in the SD medium containing 100 ppm of Abu in thesame manner as that of Example 9. As a result, it was found that theAG1-dug2Δ0 strain contained a larger amount of γ-Glu-Abu. It wassuggested that disruption of the enzyme that decomposed GSH was usefulfor accumulation of γ-Glu-Abu.

TABLE 15 Effect of addition of Abu γ-Glu-Abu γ-Glu-Abu-Gly Abu (ppm)(ppm) (ppm) AG1 strain 998 30467 1020 AG1-dug2Δ0 strain 782 39989 1746

INDUSTRIAL APPLICABILITY

According to the present invention, a yeast containing γ-Glu-Abu and ayeast extract containing γ-Glu-Abu can be produced. The yeast extractcontaining the peptide has a superior effect of imparting kokumi,especially initial taste type kokumi.

1. A yeast extract containing 0.2% or more of γ-Glu-Abu based on dryweight of the yeast extract.
 2. A yeast extract according to claim 1,which contains 0.5% or more of γ-Glu-Abu based on dry weight of theyeast extract.
 3. A yeast extract according to claim 1, which contains1.0% or more of γ-Glu-Abu based on dry weight of the yeast extract. 4.The yeast extract according to claim 1, wherein the yeast belongs to thegenus Saccharomyces or Candida.
 5. The yeast extract according to claim2, wherein the yeast belongs to the genus Saccharomyces or Candida. 6.The yeast extract according to claim 3, wherein the yeast belongs to thegenus Saccharomyces or Candida.
 7. The yeast extract according to claim4, which is obtained from Saccharomyces cervisiae or Candida utilis. 8.The yeast extract according to claim 5, which is obtained fromSaccharomyces cervisiae or Candida utilis.
 9. The yeast extractaccording to claim 6, which is obtained from Saccharomyces cervisiae orCandida utilis.
 10. A method for producing a yeast extract containingγ-Glu-Abu, which comprises culturing a yeast in a medium to which acompound selected from Abu and γ-Glu-Abu is added, and preparing a yeastextract from the obtained cells.
 11. The method according to claim 10,wherein the compound is added to the medium in an amount of 10 ppm ormore in the case of Abu, or 1 ppm or more in the case of γ-Glu-Abu, andthe yeast extract contains 0.2% or more of γ-Glu-Abu based on dry weightof the yeast extract.
 12. The method according to claim 10, wherein theyeast belongs to the genus Saccharomyces or Candida.
 13. The methodaccording to claim 11, wherein the yeast belongs to the genusSaccharomyces or Candida.
 14. The method according to claim 12, whereinthe yeast is Saccharomyces cervisiae or Candida utilis.
 15. The methodaccording to claim 13, wherein the yeast is Saccharomyces cervisiae orCandida utilis.
 16. The method according to claim 10, wherein the yeasthas one or both of the following characteristics: (a) γ-glutamylcysteinesynthetase activity is enhanced, (b) glutathione synthetase activity isattenuated.
 17. A yeast having an increased γ-Glu-Abu content, which hasbeen modified so that activity of aminotransferase or/and activity ofα-ketobutyric acid synthetase are enhanced, and, activity ofγ-glutamylcysteine synthetase is enhanced, or/and activity ofglutathione synthetase is attenuated.
 18. The yeast according to claim17, wherein the aminotransferase is an enzyme encoded by the BAT1 gene.19. The yeast according to claim 17, wherein the aminotransferase is anenzyme encoded by the UGA1 gene.
 20. The yeast according to claim 17,wherein the α-ketobutyric acid synthetase is an enzyme encoded by theCHA1 gene.
 21. The yeast according to claim 17, wherein a peptidaseactivity is further attenuated.
 22. A yeast extract produced from theyeast according to claim
 18. 23. A method for producing a yeast extractcontaining γ-Glu-Abu, which comprises allowing a γ-glutamyltransferaseto act on a yeast extract raw material to which Abu has been added. 24.The method according to claim 23, wherein Abu is added in an amount of0.1% or more based on dry weight of the yeast extract raw material, andthe yeast extract contains 0.2% or more of Abu based on dry weight ofthe yeast extract.